Methods for treating a culture of haematococcus pluvialis for lysis using hydrogen peroxide

ABSTRACT

Methods of treating lysis in cultures of  Haematococcus pluvialis  with hydrogen peroxide are described herein. The method comprises dosing the culture comprising with a concentration of hydrogen peroxide based on the stage of the cells in the culturing process and at a frequency to increase the likelihood of the cells surviving until the process of accumulating carotenoids, such as astaxanthin, is complete.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/667,917, filed Mar. 31, 2015, entitled Methods for Treating a Cultureof Haematococcus pluvialis for Contamination Using Hydrogen Peroxide,the entire contents of which are hereby incorporated by reference.

BACKGROUND

Haematococcus is a microalga that is capable of producing astaxanthin, ahigh value carotenoid with antioxidant properties. The culturing processfrom beginning to end is relatively long compared to other commonmicroalgae, such as Chlorella or Nannochloropsis, and results in anumber of challenges to the survival of Haematococcus cells due to thenature of Haematococcus as a slow growing microalga. Over the course ofthe culturing process, the Haematococcus cells must go through a growthand cell division phase to accumulate biomass before entering a secondstage where growth and motility is halted but astaxanthin is accumulatedin the cells before harvest. Operating this long multi-stage culturingprocess as an open culture increases exposure of the cells to thedangers of contamination, a sub-optimal environment, or other conditionswhich reduce the survival rate of the cells and ultimately the quantityand quality of the astaxanthin harvest.

Developing treatments for increasing the survival rate of Haematococcuscultures must take into account the sensitivities of the cells at thedifferent stages, impact on biomass growth, and impact on astaxanthinproduction, as well as effectiveness of the treatment over the longculturing process. Treatments developed for faster growing microalgae ormicroalgae cultured for production of whole biomass, lipids, orproteins, such as treatment with oxidative agents or commerciallyavailable herbicides, fungicides, and pesticides, have not been shown tobe easily translatable to Haematococcus cultures due to the uniquestages of the Haematococcus culturing process, the sensitivities ofHaematococcus cells, and the desire to use the targeted end product ofHaematococcus cultures in human consumption product industries (e.g.,nutritional supplements, food enhancers, therapeutic compositions).Therefore, there is a need in the art to development treatment methodsfor increasing the survival rate of Haematococcus cells before andduring the astaxanthin accumulation stage, without adversely affectingthe cells and value of the end product.

SUMMARY

In one non-limiting embodiment of the invention, a method of culturingHaematococcus pluvialis, may comprise: culturing a population ofHaematococcus pluvialis cells in growth conditions in a liquid culturemedium to obtain a culture of Haematococcus pluvialis cells in which thecells are primarily in the green swimmer stage; contacting the primarilygreen swimmer stage culture with hydrogen peroxide to form a calculatedconcentration in the range of 0.005-0.020 mL of hydrogen peroxide per Lof culture medium (mL/L); and culturing the Haematococcus pluvialiscells in reddening conditions to form cells in the red cyst stage foraccumulation of carotenoids.

In some embodiments, the calculated concentration of hydrogen peroxidemay be in the range of 0.005-0.010 mL/L. In some embodiments, thecalculated concentration of hydrogen peroxide may be in the range of0.010-0.015 mL/L. In some embodiments, the calculated concentration ofhydrogen peroxide is in the range of 0.015-0.020 mL/L.

In some embodiments, the growth conditions may comprise aphotosynthetically active radiation intensity in the range of 30-60 molm⁻² d⁻¹, nitrate concentration in the range of 20-50 ppm in the culturemedium, and less than 1 ppt of sodium chloride in the culture medium. Insome embodiments, the reddening conditions may comprise the present of1-5 ppt sodium chloride in the culture medium.

In some embodiments, the method may further comprise determining a levelof chytrids in the culture of Haematococcus pluvialis cells as apercentage of infected cells out of the total cells in a culture. Insome embodiments, the culture of Haematococcus pluvialis cells may becontacted with the hydrogen peroxide when the level of chytrids is lessthan 20%. In some embodiments, the culture of Haematococcus pluvialiscells is contacted with the hydrogen peroxide when the level of chytridsis at least 5%.

In some embodiments, the level of chytrids in the culture may bemaintained below the level of chytrids at the time of contact withhydrogen peroxide while culturing the Haematococcus pluvialis cells inreddening conditions to produce cells in the red cyst stage for theaccumulation of carotenoids. In some embodiments, the chytrid levelafter contacting the culture with hydrogen peroxide may be 20-95% lessthan a control culture not receiving treatment with hydrogen peroxide.

In some embodiments, the cells may be contacted with the hydrogenperoxide multiple times. In some embodiments, the cells may be contactedwith the hydrogen peroxide every 6-24 hours. In some embodiments, thecells may be contacted with the hydrogen peroxide every 6-12 hours. Insome embodiments, the cells may be contacted with the hydrogen peroxideevery day over the course of 1-14 days. In some embodiments, the cellsmay be contacted with hydrogen peroxide every other day over the courseof 3-15 days.

In some embodiments, the biomass yield of the Haematococcus pluvialiscells contacted with the hydrogen peroxide may be equivalent to orgreater than a control culture not receiving treatment with hydrogenperoxide. In some embodiments, the biomass yield of the Haematococcuspluvialis cells contacted with the hydrogen peroxide may be 0.01-0.25g/L greater than a control culture not receiving treatment with hydrogenperoxide.

In some embodiments, the carotenoids yield of the Haematococcuspluvialis cells contacted with the hydrogen peroxide may be equivalentto or greater than a control culture not receiving treatment withhydrogen peroxide. In some embodiments, the carotenoid yield of theHaematococcus pluvialis cells contacted with the hydrogen peroxide maybe 0.10-1.50% greater than a control culture not receiving treatmentwith hydrogen peroxide.

DETAILED DESCRIPTION OF THE INVENTION Overview

Haematococcus is a genus of microalgae classified in the Eukaryotadomain, Viridiplantae kingdom, Chlorophyta phylum, Chlorophyceae class,Chlamydomonadales order, and Haematococcaceae family. The speciesHaematococcus pluvialis is typically grown in phototrophic conditionsand is of particular interest commercially for the production ofastaxanthin, a high value carotenoid (i.e., organic pigment) with strongantioxidant properties. While Haematococcus pluvialis producesastaxanthin, the level of astaxanthin in the cell is dependent on theculturing conditions and is not present at a constant level in the cellover the life of the cell.

Haematococcus pluvialis has been studied academically and producedcommercially, and thus conventional culture conditions may be found inliterature in the public domain. A culture of Haematococcus pluvialiscells begins in growth conditions, where the cells are primarily (i.e.,at least 80% of cells) in the green swimmer stage in which the cells maygrow and divide but have low levels of astaxanthin. The term “greenswimmer” refers to a state of the Haematococcus pluvialis cell in whichthe cell is in a motile state, contains cilia, and has a largerproportion of chlorophyll (i.e., green pigment) than carotenoids (i.e.,red pigment from astaxanthin). Haematococcus pluvialis cells may alsoexist in a non-motile or cyst stage when the cell has a largerproportion of chlorophyll (i.e., green pigment) than carotenoids (i.e.,red pigment from astaxanthin), which may be referred to as a “greencyst”. The term “growth conditions” refers to culture conditions thatfacilitate the growth and cell division of the Haematococcus pluvialiscells, and minimize the stressors that may cause a cell to enter aresting state. Growth conditions for Haematococcus pluvialis cells maycomprise light in the photosynthetically active radiation (PAR)wavelengths, carbon dioxide, and a liquid medium comprising primarilywater, nitrogen, phosphorus and trace metal nutrients.

As Haematococcus pluvialis cells mature, they transition to a red cyststage where cell division halts or slows but astaxanthin is accumulatedas the cell is stressed by reddening conditions. The term “red cyst”refers to a state of the Haematococcus pluvialis cell in which the cellis in a resting state, has lost the cilia, and has a larger proportionof carotenoids (i.e., red pigment from astaxanthin) than chlorophyll(i.e., green pigment). The term “reddening conditions” refers to cultureconditions that stress the Haematococcus pluvialis cells to facilitatethe transition to a resting state and accumulation of carotenoids (e.g.,astaxanthin) in the cells. Reddening conditions for Haematococcuspluvialis may comprise nitrogen or other nutrient deprivation, additionof bi-carbonate, addition of bleach, and increased levels of salinity,light intensity, and/or temperature as compared to growth conditions.

Due to the size of Haematococcus cells, the culture is actively mixed bymeans known in the art such as, but not limited to, paddlewheels, gassparging, and mechanical stirrers, in order to prevent the cells fromsettling to the bottom of the bioreactor and to circulate the cells forexposure to available light and nutrients. Haematococcus pluvialis maybe cultured in a number of systems known in the art that meet the shearsensitivity requirements for green swimmer cells such as, but notlimited to, column bioreactors with gas sparger mixing, raceway pondbioreactors with paddlewheel mixing, and bag bioreactors with gassparger mixing.

The process of culturing a small volume Haematococcus culture throughthe green swimmer stage to a large volume in the red cyst stage may takeweeks due to the slow rate at which Haematococcus grows, divides, andaccumulates carotenoids. During this time period the Haematococcus cellsare vulnerable to weakening of the physical integrity of the cells(e.g., lysis) and to attacks by contamination (e.g., bacteria, fungi,predator organisms, other microalgae) which reduce the chances ofHaematococcus survival in both the green swimmer and red cyst stages.The term “lysis” refers to Haematococcus pluvialis cells losing theintegrity of the cell membrane and breaking open the outside of the haloor lysing the internal cytoplasm without halo breakage, and is expressedas a % of the total Haematococcus pluvialis cells in the culture.

For example, the occurrence of lysis in the green swimmer stage and achytrid infection in the non-motile cell stages, including green and redcyst stage, have been observed to rapidly kill the majority ofHaematococcus cells in a culture. Chytrids are a basal fungus whichoperates by attaching to microalgae cells, growing into the microalgaecell, reproducing in the cell, and subsequently attacking moremicroalgae cells. Such a loss of a Haematococcus culture after resourceshave been expended to culture the cells for multiple days or weeks, butbefore a harvest of the cells with a desirable level of astaxanthin canbe obtained, may be devastating for a commercial operation. Thevulnerability of the Haematococcus cells is further amplified in opencultures (e.g., open pond bioreactors), where conditions are harder tocontrol and contamination is more easily introduced.

The length of the culturing process for Haematococcus increases thenecessity for treatments to the culture be capable of applicationmultiple times over the course of the culturing process without harmingthe Haematococcus cells, or application of a high initial concentrationthat remains effective for a long period but does not harm theHaematococcus cells at the initial application. Treatments where theHaematococcus could not tolerate a one-time application at an initialconcentration high enough to maintain effectiveness againstcontamination over time, or where multiple applications would accumulatea concentration level toxic to the Haematococcus would not achieve thegoal of getting the culture to a successful harvest. Additionally, thesensitivity of the Haematococcus cells is dependent on the stage orstate of the cells, with the green swimmer cells being more sensitivethan the red cyst cells. For example, lysis is more likely to occurwithin a culture of green swimmer cells than in a culture of red cystcells, and green swimmer cells are less tolerant of salt than red cystcells. A general treatment may only be effective for one stage of aHaematococcus culture or may be harmful to cells in a certain state,therefore a successful treatment over the life of a Haematococcusculture must take into account the state of the cells in order tomaximize effectiveness and minimize or eliminate adverse effects on thecells.

Tests of available treatments, including chemical biocides, blends ofnatural organic herbs, bleach, sodium hydroxide, and biological agentswere found to have varying levels of effectiveness against chytrids inexamples 13-17. However, the public domain knowledge for these availabletreatments does not address how these treatments will affectHaematococcus cells in the green swimmer and red cyst stages, andtherefore do not provide immediately available solutions to thedescribed challenges faced in culturing Haematococcus.

Known methods of adaptation or genetic modification may be used to alterthe Haematococcus cells for increased resistance to lysis orcontamination, however the process may be long and expensive.Additionally, genetic modification may limit product markets availablefor using the Haematococcus derived astaxanthin.

The inventors have developed the described methods specific to the greenswimmer and cyst stages, including red cysts and green cysts, for use inthe contexts of prevention of lysis or fungal infection of aHaematococcus culture and treating a culture of Haematococcus cells withexisting levels of lysis or fungal infection. Some embodiments of themethods may be used multiple times to treat the same culture ofHaematococcus, including treating the same culture multiple times in asingle day, while minimizing or eliminating any negative effect on thebiomass yield and carotenoid yield of the cells.

The inventors surprisingly found that treatment of a culture ofHaematococcus pluvialis with hydrogen peroxide was successful inpreventing and treating lysis in a culture of Haematococcus cellswithout negatively effecting biomass accumulation and productivity ofthe cells, even when administered multiple times. The inventors alsosurprisingly found that treatments of a culture of Haematococcuspluvialis with hydrogen peroxide, salt, or hydrogen peroxide incombination with salt were successful in preventing and treating achytrid infection in a culture of Haematococcus cells without negativelyaffecting biomass accumulation, productivity, and carotenoidaccumulation, even when administered multiple times. Hydrogen peroxidewas also found to be advantageous due to the ability to dissipatequickly in the Haematococcus culture (e.g., degrades to undetectablelevels within 2 hours of application), which allows multipleapplications to be applied without the danger of buildup of residualconcentrations or detection in the final harvested product. Salt wastreatments were found to be advantageous in that the concentration ofsalt in the culture may be a result of an single dosing at a desiredconcentration or multiple doses building up to the desiredconcentration, but remain effective over time without determinant to theHaematococcus cells or harvested product.

Hydrogen peroxide may be purchased commercially at different stockconcentrations, therefore a calculated concentration was used todescribe the inventive methods. The term “calculated concentration” is aconcentration value for a contamination treatment solution calculated bymultiplying the volume of the treatment solution per culture volume(e.g., mL of hydrogen peroxide/L of microalgae culture medium) by thepercent stock concentration of the contamination treatment solution. Thecalculated concentration expressed is in units of volume/volume (e.g.,mL/L) for a theoretical 100% stock concentration of treatment solutionapplied to a microalgae culture. For example, a 1 L microalgae culturetreated with 10 mL of a hydrogen peroxide solution of a stockconcentration of 50% would have a calculated concentration=10 mL/L×0.5=5mL/L contamination treatment solution.

The described methods of applying an effective concentration of hydrogenperoxide, salt, or combination of hydrogen peroxide and salt to aculture of Haematococcus may prevent the occurrence of lysis or achytrid infection, prevent an increase in the lysis or chytrid infectionlevel (i.e., maintain the level), slow the increase of the lysis orchytrid infection level, or decrease the lysis or chytrid infectionlevel in order to increase the survival rate of the Haematococcus cellsand decrease any negative effect on the accumulation of astaxanthin. Thelevel of tolerance a culture of Haematococcus has for lysis or chytridsmay vary depending on the strain, culture conditions, and bioreactorsystem. Some cultures may be able to survive a lysis or chytridinfection level above 50%, while other cultures may only survive atlower levels such as below 30%, 20%, 10%, or 5%.

While the prior art has generally disclosed the presence of hydrogenperoxide in a microalgae or cyanobacteria culture for a variety offunctions, the teachings of the prior art relate to the use of hydrogenperoxide in contexts not directly translatable to culturingHaematococcus for astaxanthin product, such as: sterilizing bioreactorswith vapor hydrogen peroxide and culturing cyanobacteria geneticallymodified for resistance to the residual hydrogen peroxide (0.0024-1.1790mL/L) from the bioreactor sterilization step; killing bacteria in amicroalgae culture with intermittent doses of hydrogen peroxide(0.00001-0.2 mL/L, 0.0590-0.7074 mL/L); and providing lethal stressconditions for programmed death of a genetically modified microalgaeusing hydrogen peroxide (at least 0.1769 mL/L). The wide ranges ofhydrogen peroxide for different purposes disclosed in the prior art donot address effective concentrations that treat or prevent lysis andfungal infections while avoiding adverse effects on Haematococcus ingreen swimmer and red cyst cell stages. Due to this deficiency in thepublically available information, such determinations for the properconcentrations of hydrogen peroxide and methods of application forHaematococcus cells in various stages of the culturing process weredetermined through extensive experimentation of two differentHaematococcus pluvialis strains by the inventors. Additionally, theextensive experimentation by the inventors resulted in the methods ofeffectively promoting survival of Haematococcus cells through preventionand treatment of lysis and fungal infections while also not adverselyaffecting biomass accumulation, productivity, and carotenoidaccumulation of the cells, which is an additional step of commercialimportance not addressed by the prior art.

Method Embodiments

In one non-limiting embodiment, a method of preventing and/or treating achytrid infection in a culture of Haematococcus may comprise: culturinga population of Haematococcus cells in growth conditions in a liquidculture medium to obtain a culture of Haematococcus cells in which thecells are primarily (i.e., at least 80%) in the green swimmer stage;contacting the primarily green swimmer cell stage culture with aneffective amount of hydrogen peroxide; and culturing the Haematococcuscells in reddening conditions to form cells in the red cyst stage forthe accumulation of carotenoids, such as astaxanthin.

In some embodiments, the Haematococcus cells may be contacted withhydrogen peroxide to form a calculated concentration in the range of0.005-0.025 mL/L. In some embodiments, the Haematococcus cells may becontacted with hydrogen peroxide to form a calculated concentration inthe range of 0.005-0.020 mL/L. In some embodiments, the Haematococcuscells may be contacted with hydrogen peroxide to form a calculatedconcentration in the range of 0.005-0.010 mL/L. In some embodiments, theHaematococcus cells may be contacted with hydrogen peroxide to form acalculated concentration in the range of 0.010-0.015 mL/L. In someembodiments, the Haematococcus cells may be contacted with hydrogenperoxide to form a calculated concentration in the range of 0.015-0.020mL/L. In some embodiments, the Haematococcus cells may be contacted withhydrogen peroxide to form a calculated concentration in the range of0.020-0.025 mL/L.

The hydrogen peroxide may be in liquid form. In some embodiments, thehydrogen peroxide may be introduced into the culture at a location ofactive mixing such as, but not limited to at the point of paddlewheel ormechanical stirring, and a point of high turbulence caused by gassparging. In some embodiments, the hydrogen peroxide may be added all ata single point. In some embodiments, the hydrogen peroxide may be evenlyapplied over a surface area.

In some embodiments, the cells may be contacted with hydrogen peroxidemultiple times. In some embodiments, the cells may be contacted withhydrogen peroxide multiple times while the cells are in the greenswimmer stage. In some embodiments, the cells may be contacted withhydrogen peroxide multiple times while the cells are in the red cyststage. In some embodiments, the cells may be contacted with hydrogenperoxide multiple times while the cell stages span the green swimmer andred cyst stage. In some embodiments, the cells may be contacted multipletimes while the culture is in growth conditions. In some embodiments,the cells may be contacted multiple times while the culture is inreddening conditions. In some embodiments, the cells may be contactedmultiple times while the culture conditions span growth and reddeningconditions.

In some embodiments, the cells may be contacted with hydrogen peroxide2-4 times per day. In some embodiments, the cells may be contacted withhydrogen peroxide every 6-24 hours. In some embodiments, the cells maybe contacted with hydrogen peroxide every 6-8 hours. In someembodiments, the cells may be contacted with hydrogen peroxide every6-12 hours. In some embodiments, the cells may be contacted withhydrogen peroxide every 12-18 hours. In some embodiments, the cells maybe contacted with hydrogen peroxide every 18-24 hours.

In some embodiments, the cells may be contacted with hydrogen peroxideevery day over the course of 1-14 days. In some embodiments, the cellsmay be contacted with hydrogen peroxide every day over the course of 1-2days. In some embodiments, the cells may be contacted with hydrogenperoxide every day over the course of 1-3 days. In some embodiments, thecells may be contacted with hydrogen peroxide every day over the courseof 1-5 days. In some embodiments, the cells may be contacted withhydrogen peroxide every day over the course of 5-7 days. In someembodiments, the cells may be contacted with hydrogen peroxide every dayover the course of 7-10 days. In some embodiments, the cells may becontacted with hydrogen peroxide every day over the course of 10-12days. In some embodiments, the cells may be contacted with hydrogenperoxide every day over the course of 12-14 days.

In some embodiments, the cells may be contacted with hydrogen peroxideevery 24-72 hours. In some embodiments, the cells may be contacted withhydrogen peroxide every 36-60 hours. In some embodiments, the cells maybe contacted with hydrogen peroxide every 42-54 hours. In someembodiments, the cells may be contacted with hydrogen peroxide everyother day over the course of 3-15 days. In some embodiments, the cellsmay be contacted with hydrogen peroxide every other day over the courseof 3-5 days. In some embodiments, the cells may be contacted withhydrogen peroxide every other day over the course of 5-7 days. In someembodiments, the cells may be contacted with hydrogen peroxide everyother day over the course of 7-9 days. In some embodiments, the cellsmay be contacted with hydrogen peroxide every other day over the courseof 9-11 days. In some embodiments, the cells may be contacted withhydrogen peroxide every other day over the course of 11-13 days. In someembodiments, the cells may be contacted with hydrogen peroxide everyother day over the course of 13-15 days.

In some embodiments, the cells may contacted with hydrogen peroxide in acombination of days over an extended time period, such as every day fora period of days and then every other day for a period of days, or visversa. For example, the cells may be contacted with hydrogen peroxideevery day for a period of 1-5 days and then every other day for a periodof 3-15 days. In other embodiments, the cells may contacted withhydrogen peroxide with more than one day in between applications, suchas but not limited to, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,or more between applications. In some embodiments, an application ofhydrogen peroxide at a scheduled time (e.g., after 6 hours, after 24hours) may be skipped and application may be resumed at a later time.

In some embodiments, the growth conditions for a culture ofHaematococcus may comprise a photosynthetically active radiation (PAR)intensity in the range of 30-60 mol m⁻² d⁻¹. In some embodiments, thegrowth conditions for a culture of Haematococcus may comprise a nitrateconcentration in the range of 20-50 ppm. In some embodiments, the growthconditions for a culture of Haematococcus may comprise a concentrationof less than 1 ppt salt, such as sodium chloride, in the culture medium.

In some embodiments, the reddening conditions for a culture ofHaematococcus may comprise the presence of salt in the culture medium.In some embodiments, the concentration of sodium chloride in thereddening conditions may comprise 1-5 ppt. In some embodiments, theconcentration of sodium chloride in the reddening conditions maycomprise 1-2 ppt. In some embodiments, the concentration of sodiumchloride in the reddening conditions may comprise 1-3 ppt. The salt maycomprise but is not limited to sodium chloride.

In some embodiments, a level of chytrids or chytrid infection in aculture of Haematococcus may be determined and expressed as a percentageof infected cells out of the total cells in a culture. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 60%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 50%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 40%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 30%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 20%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 10%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 5%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 3%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 2%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is less than 1%. In someembodiments, the culture of Haematococcus cells may be contacted withhydrogen peroxide when the level of chytrids is 0%.

In some embodiments, the culture of Haematococcus cells may be contactedwith hydrogen peroxide when the level of chytrids is at least 1%. Insome embodiments, the culture of Haematococcus cells may be contactedwith hydrogen peroxide when the level of chytrids is at least 2%. Insome embodiments, the culture of Haematococcus cells may be contactedwith hydrogen peroxide when the level of chytrids is at least 5%. Insome embodiments, the culture of Haematococcus cells may be contactedwith hydrogen peroxide when the level of chytrids is at least 10%. Insome embodiments, the culture of Haematococcus cells may be contactedwith hydrogen peroxide when the level of chytrids is at least 20%. Insome embodiments, the culture of Haematococcus cells may be contactedwith hydrogen peroxide when the level of chytrids is at least 30%. Insome embodiments, the culture of Haematococcus cells may be contactedwith hydrogen peroxide when the level of chytrids is at least 40%. Insome embodiments, the culture of Haematococcus cells may be contactedwith hydrogen peroxide when the level of chytrids is at least 50%. Insome embodiments, the level of chytrids in a culture of Haematococcuscells may be maintained below the level of chytrids at the time ofcontact with hydrogen peroxide while culturing the Haematococcus cellsin reddening conditions to form cells in the red cyst stage for theaccumulation of carotenoids.

In some embodiments, the chytrid level after contacting the culture withhydrogen peroxide may be less than a control culture not receivingtreatment with hydrogen peroxide. In some embodiments, the chytrid levelafter contacting the culture with hydrogen peroxide may be reduced by20-95% compared to a control culture not receiving treatment withhydrogen peroxide. In some embodiments, the chytrid level aftercontacting the culture with hydrogen peroxide may be reduced by 20-30%compared to a control culture not receiving treatment with hydrogenperoxide. In some embodiments, the chytrid level after contacting theculture with hydrogen peroxide may be reduced by 30-40% compared to acontrol culture not receiving treatment with hydrogen peroxide. In someembodiments, the chytrid level after contacting the culture withhydrogen peroxide may be reduced by 40-50% compared to a control culturenot receiving treatment with hydrogen peroxide. In some embodiments, thechytrid level after contacting the culture with hydrogen peroxide may bereduced by 50-60% compared to a control culture not receiving treatmentwith hydrogen peroxide. In some embodiments, the chytrid level aftercontacting the culture with hydrogen peroxide may be reduced by 60-70%compared to a control culture not receiving treatment with hydrogenperoxide. In some embodiments, the chytrid level after contacting theculture with hydrogen peroxide may be reduced by 70-80% compared to acontrol culture not receiving treatment with hydrogen peroxide. In someembodiments, the chytrid level after contacting the culture withhydrogen peroxide may be reduced by 80-90% compared to a control culturenot receiving treatment with hydrogen peroxide. In some embodiments, thechytrid level after contacting the culture with hydrogen peroxide may bereduced by 90-95% compared to a control culture not receiving treatmentwith hydrogen peroxide.

In some embodiments, the chytrid level after contacting the culture withhydrogen peroxide in combination with salt may be less than a controlculture not receiving treatment. In some embodiments, the chytrid levelafter contacting the culture with hydrogen peroxide in combination withsalt may be reduced by 10-95% compared to a control culture notreceiving treatment. In some embodiments, the chytrid level aftercontacting the culture with hydrogen peroxide in combination with saltmay be reduced by 10-30% compared to a control culture not receivingtreatment. In some embodiments, the chytrid level after contacting theculture with hydrogen peroxide in combination with salt may be reducedby 30-60% compared to a control culture not receiving treatment. In someembodiments, the chytrid level after contacting the culture withhydrogen peroxide in combination with salt may be reduced by 60-95%compared to a control culture not receiving treatment.

In some embodiments, the biomass yield of the Haematococcus cellscontacted with hydrogen peroxide is equivalent to or greater than acontrol culture not receiving treatment with hydrogen peroxide. In someembodiments, the biomass yield of the Haematococcus cells contacted withhydrogen peroxide is 0.01-0.25 g/L greater than a control culture notreceiving treatment with hydrogen peroxide. In some embodiments, thebiomass yield of the Haematococcus cells contacted with hydrogenperoxide is 0.01-0.05 g/L greater than a control culture not receivingtreatment with hydrogen peroxide. In some embodiments, the biomass yieldof the Haematococcus cells contacted with hydrogen peroxide is 0.05-0.10g/L greater than a control culture not receiving treatment with hydrogenperoxide. In some embodiments, the biomass yield of the Haematococcuscells contacted with hydrogen peroxide is 0.10-0.15 g/L greater than acontrol culture not receiving treatment with hydrogen peroxide. In someembodiments, the biomass yield of the Haematococcus cells contacted withhydrogen peroxide is 0.15-0.20 g/L greater than a control culture notreceiving treatment with hydrogen peroxide. In some embodiments, thebiomass yield of the Haematococcus cells contacted with hydrogenperoxide is 0.20-0.25 g/L greater than a control culture not receivingtreatment with hydrogen peroxide.

In some embodiments, the biomass yield of the Haematococcus cellscontacted with hydrogen peroxide in combination with salt is equivalentto or greater than a control culture not receiving treatment. In someembodiments, the biomass yield of the Haematococcus cells contacted withhydrogen peroxide in combination with salt is 0.01-0.30 g/L greater thana control culture not receiving treatment. In some embodiments, thebiomass yield of the Haematococcus cells contacted with hydrogenperoxide in combination with salt is 0.01-0.10 g/L greater than acontrol culture not receiving treatment. In some embodiments, thebiomass yield of the Haematococcus cells contacted with hydrogenperoxide in combination with salt is 0.10-0.20 g/L greater than acontrol culture not receiving treatment. In some embodiments, thebiomass yield of the Haematococcus cells contacted with hydrogenperoxide in combination with salt is 0.20-0.30 g/L greater than acontrol culture not receiving treatment.

In some embodiments, the carotenoid yield of the Haematococcus cellscontacted with hydrogen peroxide is equivalent to or greater than acontrol culture not receiving treatment with hydrogen peroxide. In someembodiments, the carotenoid yield of the Haematococcus cells contactedwith hydrogen peroxide is 0.10-1.50% greater than a control culture notreceiving treatment with hydrogen peroxide. In some embodiments, thecarotenoid yield of the Haematococcus cells contacted with hydrogenperoxide is 0.10-0.25% greater than a control culture not receivingtreatment with hydrogen peroxide. In some embodiments, the carotenoidyield of the Haematococcus cells contacted with hydrogen peroxide is0.25-0.50% greater than a control culture not receiving treatment withhydrogen peroxide. In some embodiments, the carotenoid yield of theHaematococcus cells contacted with hydrogen peroxide is 0.50-0.75%greater than a control culture not receiving treatment with hydrogenperoxide. In some embodiments, the carotenoid yield of the Haematococcuscells contacted with hydrogen peroxide is 0.75-1.00% greater than acontrol culture not receiving treatment with hydrogen peroxide. In someembodiments, the carotenoid yield of the Haematococcus cells contactedwith hydrogen peroxide is 1.00-1.25% greater than a control culture notreceiving treatment with hydrogen peroxide. In some embodiments, thecarotenoid yield of the Haematococcus cells contacted with hydrogenperoxide is 1.25-1.50% greater than a control culture not receivingtreatment with hydrogen peroxide.

In some embodiments, the method may further comprise transferring theculture of Haematococcus cells to a new culturing vessel aftercontacting the culture with the hydrogen peroxide. In some embodiments,the culture may be contacted with hydrogen peroxide when the culture isat an optimal temperature. In some embodiments, the culture may becontacted with hydrogen peroxide when the culture is at an optimalculture density.

In another non-limiting embodiment, a method of preventing and/ortreating a chytrid infection in a culture of Haematococcus may comprise:culturing a population of Haematococcus cells in reddening conditions ina liquid culture medium comprising 1-5 ppt of salt to obtain a cultureof Haematococcus cells in which the cells are primarily (i.e., at least80%) in the red cyst stage for the accumulation of carotenoids, such asastaxanthin; and contacting the primarily red cyst cell stage culturewith an effective amount of hydrogen peroxide. In some embodiments, theHaematococcus cells in which the cells may be primarily (i.e., at least80%) in the green cyst stage in reddening conditions, a combination ofgreen and red cysts in reddening conditions, or a non-motile state inreddening conditions when contacted with an effective amount of hydrogenperoxide.

In another non-limiting embodiment, a method of treating a chytridinfection in a culture of Haematococcus may comprise: culturing apopulation of Haematococcus cells in reddening conditions in a liquidculture medium to obtain a culture of Haematococcus cells in which thecells are primarily (i.e., at least 80%) in the red cyst stage for theaccumulation of carotenoids, such as astaxanthin; detecting a presenceof chytrids in the culture; and contacting the culture comprisingchytrids and primarily red cyst cells with an effective amount of salt.In some embodiments, the Haematococcus cells in which the cells may beprimarily (i.e., at least 80%) in the green cyst stage in reddeningconditions, a combination of green and red cysts in reddeningconditions, or a non-motile state in reddening conditions when contactedwith an effective amount of salt.

In some embodiments, the salt contacting the chytrids and red cyst cellsmay be sodium chloride. In some embodiments, the effective amount ofsodium chloride is at least 1.5 times the amount of sodium chloridefound in typical reddening conditions, and may be up to 10 times. Insome embodiments, the concentration of sodium chloride contacting thechytrids and red cyst cells may be 1-20 ppt. In some embodiments, theconcentration of sodium chloride contacting the chytrids and red cystcells may be 1-2 ppt. In some embodiments, the concentration of sodiumchloride contacting the chytrids and red cyst cells may be 1-3 ppt. Insome embodiments, the concentration of sodium chloride contacting thechytrids and red cyst cells may be 1-5 ppt. In some embodiments, theconcentration of sodium chloride contacting the chytrids and red cystcells may be 5-10 ppt. In some embodiments, the concentration of sodiumchloride contacting the chytrids and red cyst cells may be 10-15 ppt. Insome embodiments, the concentration of sodium chloride contacting thechytrids and red cyst cells may be 15-20 ppt.

In some embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 60%.In some embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 50%.In some embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 40%.In some embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 30%.In some embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 20%.In some embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 10%.In some embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 5%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 4%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 3%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 2%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is less than 1%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is 0%. In someembodiments, the level of chytrids in the culture may be maintainedbelow the level of chytrids at the time of contact with the salt whileculturing the Haematococcus cells in reddening conditions to form cellsin the red cyst stage for the accumulation of carotenoids.

In some embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is at least 1%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is at least 2%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is at least 5%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is at least 10%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is at least 20%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is at least 30%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is at least 40%. Insome embodiments, the culture of Haematococcus cells may be contactedwith salt when a level of cells infected by chytrids is at least 50%.

In another non-limiting embodiment, a method of preventing a chytridinfection in a culture of Haematococcus may comprise: culturing apopulation of Haematococcus cells in a liquid culture medium;determining a number of Haematococcus cells infected with chytrids inthe culture; contacting the culture with an effective amount of hydrogenperoxide when the percentage of Haematococcus cells infected withchytrids is less than a threshold level of the total cells; continuingto culture the Haematococcus cells; and verifying that a percentage ofHaematococcus cells infected with chytrids is less than a thresholdlevel of the total cells after contact with the hydrogen peroxide.

In some embodiments, the threshold level of cells infected with chytridsmay be 1% of the total cells. In some embodiments, the threshold levelof cells infected with chytrids may be 2% of the total cells. In someembodiments, the threshold level of cells infected with chytrids may be3% of the total cells. In some embodiments, the threshold level of cellsinfected with chytrids may be 4% of the total cells. In someembodiments, the threshold level of cells infected with chytrids may be5% of the total cells. In some embodiments, the threshold level of cellsinfected with chytrids may be 10% of the total cells. In someembodiments, the threshold level of cells infected with chytrids may be15% of the total cells. In some embodiments, the threshold level ofcells infected with chytrids may be 20% of the total cells. In someembodiments, the threshold level of cells infected with chytrids may be25% of the total cells. In some embodiments, the threshold level ofcells infected with chytrids may be 30% of the total cells.

In another non-limiting embodiment, a method of preventing and/ortreating lysis in a culture of Haematococcus may comprise: culturing apopulation of Haematococcus cells in a liquid culture medium in growthconditions to obtain a culture of Haematococcus cells in which the cellsare primarily (i.e., at least 80%) in the green swimmer stage;contacting the primarily green swimmer cell stage culture with aneffective amount of hydrogen peroxide prior to the formation of cellcysts; and continuing to culture the Haematococcus cells in growthconditions.

In some embodiments, the method may further comprise determining a levelof lysis in the culture of Haematococcus cells as a percentage of thetotal cells in the culture. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 30%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 25%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 20%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 15%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 10%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 5%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 4%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 3%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 2%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is less than 1%. In some embodiments, the culture ofHaematococcus cells may be contacted with hydrogen peroxide when thelevel of lysis is 0%.

In some embodiments, the level of lysis in the culture may be maintainedat or below the level of lysis at the time of contact with hydrogenperoxide while continuing to culture the Haematococcus cells in growthconditions. In some embodiments, the lysis level of the culture aftercontact with hydrogen peroxide may be 1-80% less than a lysis level in acontrol culture not receiving treatment with hydrogen peroxide. In someembodiments, the lysis level of the culture after contact with hydrogenperoxide may be 1-3% less than a lysis level in a control culture notreceiving treatment with hydrogen peroxide. In some embodiments, thelysis level of the culture after contact with hydrogen peroxide may be3-6% less than a lysis level in a control culture not receivingtreatment with hydrogen peroxide. In some embodiments, the lysis levelof the culture after contact with hydrogen peroxide may be 6-10% lessthan a lysis level in a control culture not receiving treatment withhydrogen peroxide. In some embodiments, the lysis level of the cultureafter contact with hydrogen peroxide may be 10-20% less than a lysislevel in a control culture not receiving treatment with hydrogenperoxide. In some embodiments, the lysis level of the culture aftercontact with hydrogen peroxide may be 20-40% less than a lysis level ina control culture not receiving treatment with hydrogen peroxide. Insome embodiments, the lysis level of the culture after contact withhydrogen peroxide may be 40-60% less than a lysis level in a controlculture not receiving treatment with hydrogen peroxide. In someembodiments, the lysis level of the culture after contact with hydrogenperoxide may be 60-80% less than a lysis level in a control culture notreceiving treatment with hydrogen peroxide.

In some embodiments, the method may comprise determining a live bacteriacount in the culture of Haematococcus cells. In some embodiments, thelive bacteria count may be reduced by 10-25×10⁵ CFU/mL after contactwith the hydrogen peroxide. In some embodiments, the live bacteria countmay be reduced by 10-15×10⁵ CFU/mL after contact with the hydrogenperoxide. In some embodiments, the live bacteria count may be reduced by15-20×10⁵ CFU/mL after contact with the hydrogen peroxide. In someembodiments, the live bacteria count may be reduced by 20-25×10⁵ CFU/mLafter contact with the hydrogen peroxide. In some embodiments, the livebacteria count may be maintained below 10⁷ CFU/mL following contact withthe hydrogen peroxide.

In another non-limiting embodiment, a method of preventing lysis in aculture of Haematococcus may comprise: culturing a population ofHaematococcus cells in a liquid culture medium in growth conditions toobtain a culture of Haematococcus cells in which the cells are primarily(i.e., at least 80%) in the green swimmer stage; determining a level ofcell lysis for the Haematococcus cells; contacting the primarily greenswimmer cell stage culture with an effective amount of hydrogen peroxideprior to the formation of cell cysts when the lysis level of theHaematococcus cells is less than a threshold level; continuing toculture the Haematococcus cells in growth conditions; and verifying thatthe level of lysis of Haematococcus cells is less than the thresholdlevel after contact with the hydrogen peroxide.

In some embodiments, the threshold lysis level may be 1% of the totalcells. In some embodiments, the threshold lysis level may be 2% of thetotal cells. In some embodiments, the threshold lysis level may be 3% ofthe total cells. In some embodiments, the threshold lysis level may be4% of the total cells. In some embodiments, the threshold lysis levelmay be 5% of the total cells. In some embodiments, the threshold lysislevel may be 10% of the total cells. In some embodiments, the thresholdlysis level may be 15% of the total cells. In some embodiments, thethreshold lysis level may be 20% of the total cells. In someembodiments, the threshold lysis level may be 25% of the total cells. Insome embodiments, the threshold lysis level may be 30% of the totalcells.

In some embodiments, the described methods may be applied to an openculture of Haematococcus. In some embodiments, the described methods maybe applied to an outdoor culture of Haematococcus. In some embodiments,the described methods may be applied to a closed culture ofHaematococcus. In some embodiments, the described methods may be appliedto an indoor culture of Haematococcus.

The use of hydrogen peroxide and/or salt in the described methods doesnot function as a further stress to the Haematococcus cells for theaccumulation of astaxanthin, but rather provides the function ofprevention and treatment of lysis and chytrid infections to increase thesurvival of the Haematococcus cells. The application of the describedmethods to a culture of Haematococcus in a batch process also does notprovide the necessary time period to adapt the Haematococcus cells forincreased resistance to lysis or fungal infections, which would requiremany applications over multiple generations coupled with selection ofthe positively performing cells.

The level of lysis, level of chytrid infection, and stage of theHaematococcus cells may be assessed by means known in the art such as,but not limited to, visual observation under a microscope, or automatedmonitoring with cameras and visual recognition software, spectrometers,or fluorimeters. The monitoring and detection of the Haematococcusculture, whether manual or automated, may be used to determine when thehydrogen peroxide and/or salt of the described methods is administeredto a culture by manual means, automated means, and combinations thereof.Automated monitoring and detection data may also be recorded or utilizedby a programmable logic controller to control the application ofhydrogen peroxide and/or salt to a culture of cells. Visual observationunder a microscope of the Haematococcus cultures using the describedmethods in tests also showed that the methods aided in breaking upfilamentous fungus and lowering the background bacteria population ofthe culture, which may contribute to lysis.

In some embodiments, a microalgae culture composition may comprise: apopulation of Haematococcus cells in a liquid culture medium; and acalculated concentration of hydrogen peroxide in the range of0.005-0.025 mL of hydrogen peroxide per L of culture medium (mL/L),wherein the hydrogen peroxide has been added to the culture medium inthe previous 120 minutes. In further embodiments, the culture maycomprise a concentration of 1-20 ppt of sodium chloride. In someembodiments, the Haematococcus cells of the culture composition may beprimarily (i.e., at least 80%) in the green swimmer stage. In someembodiments, the Haematococcus cells of the culture composition may beprimarily (i.e., at least 80%) in the red cyst stage.

EXAMPLES

Embodiments of the invention are exemplified and additional embodimentsare disclosed in further detail in the following Examples, which are notin any way intended to limit the scope of any aspects of the inventiondescribed herein. Within these Examples two different strains ofHaematococcus pluvialis were tested and are identified as “Strain 1” and“Strain 2”, respectively.

Example 1

Experiments were conducted to determine the degradation rate of hydrogenperoxide in a culture of Haematococcus pluvialis. An Amplex Red HydrogenPeroxide Kit (CAT. No. A22188), commercially available from LifeTechnologies (Grand Island, N.Y.), was used to assay hydrogen peroxideconcentrations according to the detection of a fluorescent productformed in the oxidation of a reagent in the presence of hydrogenperoxide. Samples of Haematococcus pluvialis (Strain 1) were taken fromcultures in a carboy bioreactor (axenic conditions) and an open racewaypond bioreactor disposed in a greenhouse with paddlewheel mixing(non-axenic conditions). Samples from both cultures were dosed with 0.06mL/L of hydrogen peroxide 25% stock concentration (effectiveconcentration of 0.015 mL/L), and the hydrogen peroxide concentrationwas monitored every 30 minutes for 180 minutes using the Amplex RedHydrogen Peroxide Kit. Samples from both cultures showed an exponentialdecay in the concentration of hydrogen peroxide, resulting in aconcentration below a level that is expected to be effective againstuniflagellates (e.g., fungi zoospores) [namely greater than 0.03 mL/L of25% stock (calculated concentration greater than 0.0075 mL/L)] after 30minutes, and an concentration of below detectable limits in 120 minutes.These results show that treating a culture with an calculatedconcentration of hydrogen peroxide of 0.015 mL/L will maintain aconcentration above the levels expected to be effective againstcontamination (e.g., uniflagellates) in the short term (i.e., <30minutes) and will thereafter dissipate to levels that are not harmful toany of the desired microorganisms in the culture, including theHaematococcus cells. The results also demonstrate that repeated doses ofhydrogen peroxide over time do not create the risk of building up aresidual concentration that would be harmful to the Haematococcus ordetectable in the harvested end product.

Example 2

Experiments were conducted to evaluate the level of hydrogen peroxidetolerance of Haematococcus pluvialis. Green swimmer cells of a firststrain of Haematococcus pluvialis (Strain 1) were tested in well platesby adding a single dose (0.06, 0.10, 0.13, 0.16 mL/L) of hydrogenperoxide 25% stock concentration to 2 mL cultures of cells. Thecalculated concentrations of the hydrogen peroxide doses tested were0.0150, 0.0250, 0.0325, and 0.0400 mL/L. The amount of cell lysis wasquantified using visual observation under a microscope 18 hours afteradministration of hydrogen peroxide. The results are presented in Table1, with standard error denoted as “SE”.

TABLE 1 H₂O₂ dosage calculated concen- % Cell Lysis tration at 18 hours(mL/L) (±1SE) 0.00 0.8% ± 0.3% (control) 0.0150 1.6% ± 1%  0.0250 1.9% ±0.2% 0.0325   2% ± 0.07% 0.0400  5.2% ± 0.48%

As shown in Table 1, significant lysis (i.e., more than 5%) of the cellsoccurred at hydrogen peroxide dosage levels above 0.0325 mL/L calculatedconcentration.

Green swimmer and red cyst cells of a second strain of Haematococcuspluvialis (Strain 2) were tested in well plates by adding a dose (0.03,0.04, 0.05, 0.06, 0.07 mL/L) of hydrogen peroxide 35% stockconcentration to 2 mL cultures of cells of both green swimmer cells andred cyst cells three times per day (i.e., morning, noon and evening).The calculated concentrations of the hydrogen peroxide doses tested were0.0105, 0.0140, 0.0175, 0.0210, and 0.0245 mL/L. The amount of celllysis was quantified using visual observation under a microscope after24, 48, and 72 hours. The results are presented in Tables 2-3, withstandard error denoted as “SE”.

TABLE 2 Green Swimmer Cells H₂O₂ dosage calculated concen- tration %Cell lysis at time periods (h) after dosing (±1SE) (mL/L) 24 48 72 0.003.3% ± 1.7% 5.0% ± 1.7%  5.0% ± 0.0% (control) 0.0105 3.3% ± 1.7% 5.0% ±1.7%  3.3% ± 0.0% 0.0140 4.2% ± 0.8% 5.8% ± 2.5%  9.2% ± 4.2% 0.01752.5% ± 0.8% 9.2% ± 2.5% 13.3% ± 3.3% 0.0210 4.2% ± 2.5% 6.7% ± 1.7%20.0% ± 3.3% 0.0245 15.0% ± 0.0%  22.5% ± 0.8%  27.5% ± 2.5%

TABLE 3 Red Cyst Cells H₂O₂ dosage calculated concen- tration % Celllysis at time periods (h) after dosing (±1SE) (mL/L) 24 48 72 96 0.001.7% ± 1.0% 0.0% ± 0.0% 0.8% ± 0.8% 2.5% ± 0.8% (control) 0.0140 1.7% ±1.0% 0.8% ± 0.8% 3.3% ± 0.0% 3.3% ± 1.7% 0.0175 1.1% ± 0.6% 3.3% ± 1.7%5.8% ± 5.8% 5.8% ± 2.5% 0.0210 3.9% ± 2.0% 0.8% ± 0.8% 1.7% ± 0.0% 18.3%± 1.7%  0.0245 3.9% ± 0.6% 5.0% ± 1.7% 9.2% ± 0.8% 51.7% ± 5.0% 

As shown in Table 2, the green swimmer cells maintained a cell lysislevel below 10% when dosed with hydrogen peroxide at a 0.0105 mL/Lcalculated concentration for at least 72 hours. At a calculatedconcentration of 0.0210 mL/L the green swimmer cells maintained lysislevels below 10% for 48 hours. These results show that the cells treatedwith lower concentration doses experienced low levels of lysis (i.e.,below 10%) for all time periods, but the cultures receiving higherconcentration doses only maintained low levels of lysis (i.e., below10%) for 48 hours or less, thus indicating that the concentration ofhydrogen peroxide in the method is critical and simply adding more doesnot equate to better results regarding lysis in green swimmer cells.

As shown in Table 3, the red cyst cells maintained a cell lysis levelbelow 10% at dosages of hydrogen peroxide below 0.0175 mL/L calculatedconcentration for at least 96 hours, but dosages at or above 0.0175 mL/Lcalculated concentration experienced cell lysis above 10% after 48-96hours. These results also show that simply increasing the dosages ofhydrogen peroxide does not produce better results with regards to lysisin red cyst cells, however the results were not exactly the same for theconcentrations when applied to green swimmer and red cyst cells.Therefore, the results of Table 2 and Table 3 together demonstrate thestate of the cell, concentration of hydrogen peroxide, and the length oftime the treatment is applied are factors that should be considered whenhydrogen peroxide is used to treat a culture of Haematococcus pluvialisto maintain cell lysis at acceptable levels or prevent lysis.

Example 3

A series of Haematococcus pluvialis (Strain 1) cultures were comparedusing bacterial community sequencing (SSU rRNA 16s) to analyze thebacterial community of the cultures during a lysis event, a chytridinfection, and during treatment with hydrogen peroxide to determine ifthe bacteria community shifts or produces a detectable pattern.

Haematococcus pluvialis cultures containing green swimmer cells in openraceway pond bioreactors [identification numbers (#) 2210 and 2220]disposed in a greenhouse, using paddlewheel mixing, and operating inconditions: reactor volume of 16,000 L; Daily photosynthetically activeradiation (PAR) of 57 mol m⁻² d⁻¹; pH of 7.5; and paddlewheel speed of40%; were inoculated on the same day and cultured in growth conditions.The culture in bioreactor #2210 was not treated with hydrogen peroxidebefore or after transfer to bioreactor #2310. The culture in thebioreactor #2220 was treated every 24 hours with 0.03 mL/L of hydrogenperoxide 25% stock concentration (calculated concentration of 0.0075mL/L) before and after the culture was transferred to open raceway pondbioreactor #2320 operating in conditions: reactor volume of 50,000 L;Daily PAR of 54 mol m⁻² d⁻¹; pH of 7.5; and paddlewheel speed of 40%;which was also disposed in a greenhouse and used paddlewheel mixing. Theresults showed that the treated culture in bioreactor #2220 retainedhigh motility (95%) one day longer than the untreated culture inbioreactor #2210, and chytrids were detected three days later in thetreated culture in bioreactor #2320 than they did in the untreatedculture of bioreactor #2310.

Nitrogen fixing bacteria including Rhizobium, Emticicia, andSinorhizobium, were identified as dominant species in the bacterialcommunity analysis of the cultures and were present in the beginning andmiddle of the culture period for each culture. However, the relativeamount of nitrogen fixing bacteria decreased from 20-40% to less than10% of the dominant bacterial community, after the hydrogen peroxidetreatments in bioreactor #2320. High amounts of cell lysis were visuallyobserved in the treated culture (bioreactor #2320), but not until fivedays after the treatment ended. Runella was dominant in the bacterialcommunity of the culture in bioreactor #2320 starting before the secondhydrogen peroxide treatment and during the period of the lysis event.

A Haematococcus pluvialis culture containing green swimmer cells in anopen raceway pond bioreactor #2330 disposed in a greenhouse, usingpaddlewheel mixing, and operating in conditions: reactor volume of55,000 L; Daily PAR of 55 mol m⁻² d⁻¹; pH of 7.5; and paddlewheel speedof 40%; was treated once with 0.03 mL/L hydrogen peroxide 25% stockconcentration (calculated concentration of 0.0075 mL/L) two days beforetransfer and twice after transfer to open raceway bioreactor #2430,which was disposed in a greenhouse, using paddlewheel mixing, andoperating in conditions: reactor volume of 140,000 L; Daily PAR of 55mol m⁻² d⁻¹; pH of 7.3; and paddlewheel speed of 70%. High amounts ofcell lysis were visually observed before the first treatment andremained high for 4 days. The effect of the hydrogen peroxide treatmentto prevent or decrease lysis appeared to be limited due to the fact thattreatment began after the lysis had occurred. Chytrid sporangia were notdetected in the culture. Rheinheirmera, Flectobacillus, Runella, andFlavobacterium, began increasing in relative to other bacteria in thebacterial community analysis of the culture during the start of lysis,however only Runella became dominant towards the end of the lysisperiod.

Another Haematococcus pluvialis culture containing green swimmer cellsin open raceway pond bioreactor #2330 operating in conditions: reactorvolume of 60,000 L; Daily PAR of 57 mol m⁻² d⁻¹; pH of 7.5; andpaddlewheel speed of 40%; was treated twice with 0.03 mL/L hydrogenperoxide 25% stock concentration (calculated concentration of 0.0075mL/L) before transfer to another bioreactor. The culture of bioreactor#2330 was transferred to open raceway pond bioreactor #2420 disposed ina greenhouse, using paddlewheel mixing, and operating in conditions:reactor volume of 140,000 L; Daily PAR of 58 mol m⁻² d⁻¹; pH of 7.3; andpaddlewheel speed of 70%); but the culture was not treated after thetransfer. This culture was observed to continue cell division aftertransfer to bioreactor #2420. Observations showed that motilitydecreased by about 30% after the first treatment and 60% lysis occurredafter the second treatment. Chytrid sporangia were observed to bepresent at the end of the culture period, 7 days after hydrogen peroxidetreatment ended. Bacteria including Pseudomonas, Flectobacillus, andCytophaga were present in the bacterial community in both thebioreactors #2330 and #2420. The percentage of carotenoids in theculture was approximately 3.5%, quantified by UV method.

Example 4

An experiment was conducted to determine if treating a culture ofHaematococcus pluvialis with hydrogen peroxide affects the live bacteriacount. Cultures of Haematococcus pluvialis (Strain 1) containing greenswimmer cells in open raceway ponds (250 L volume) with paddlewheelmixing and disposed in a warehouse were split into open pond bioreactors#2210 and 2230 disposed inside a greenhouse, using paddlewheel mixing,and operating in conditions: reactor volume of 16,000 L; Daily PAR of 50mol m⁻² d⁻¹; pH of 7.5; and paddlewheel speed of 40%. The culture inbioreactor #2210 was treated with 0.33 mL/L hydrogen peroxide 3% stockconcentration (calculated concentration of 0.0099 mL/L) every 6 hoursfor the duration of the culture's green swimmer stage (90 hours). Theculture in bioreactor #2230 was not treated to serve as a control forcomparison. Samples were taken to assess motility, lysis, and the livebacteria count in the cultures at 11, 34, 52, and 66 hours. The livebacteria count was obtained by plate count using Petrifilm availablefrom 3M (St. Paul, Minn.), and the results are shown in Table 4.

TABLE 4 Live Bacteria Count (CFU/mL BacT) Time Untreated H₂O₂ (h)Control treatment 11 3.7 × 10⁷ 4.8 × 10⁵ 34 1.0 × 10⁶ 5.5 × 10⁶ 52 1.5 ×10⁶ 1.8 × 10⁵ 66 2.9 × 10⁶ 4.5 × 10⁵ 90 8.1 × 10⁵ No data

Motility was maintained above 60% for the entire experiment but wasslightly higher in the untreated control culture. Lysis greater than 15%did not occur in either culture. As shown in Table 4, the culture thatreceived the hydrogen peroxide treatment had an approximately 1 logreduction in the bacteria count. This result may be useful in analyzingthe potential for lysis in the culture, as findings from previousexperiments showed a correlation between bacteria concentrations above10⁷ cells/mL and the occurrence of lysis in cultures of Haematococcuspluvialis. Therefore, reducing the live bacteria count of aHaematococcus pluvialis culture with a hydrogen peroxide treatment canbe used to prevent conditions that are favorable for lysis and reducethe risk of losing Haematococcus pluvialis cells to lysis.

Example 5

A series of experiments were conducted to determine if treating aculture of Haematococcus pluvialis with hydrogen peroxide preventslysis. Samples of a culture of Haematococcus pluvialis (Strain 1)containing green swimmer cells were taken from open raceway pondbioreactor #2330 disposed in a greenhouse, using paddlewheel mixing, andoperating in conditions: reactor volume of 60,000 L; Daily PAR of 57 molm⁻² d⁻¹; pH of 7.5; and paddlewheel speed of 40%; before the culture wastransferred to open raceway pond bioreactor #2430 disposed in agreenhouse, using paddlewheel mixing, and operating in conditions:reactor volume of 140,000 L; Daily PAR of 58 mol m⁻² d⁻¹; pH of 7.3; andpaddlewheel speed of 70%); as well as 24 and 48 hours after transfer tobioreactor #2430. The samples were divided into flasks with some flasksreceiving treatment with 0.028 mL/L hydrogen peroxide 25% stockconcentration (calculated concentration of 0.007 mL/L) three times perday, and some flasks receiving no treatment to serve as controls forcomparison. Lysis was quantified daily through visual observation ofsamples under a microscope. The results are shown in Tables 5-7, withstandard deviation denoted as “SD”.

TABLE 5 % Lysis in samples from Bioreactor # 2330 (±1SD) Time UntreatedH₂O₂ (h) Control Treatment 0 8.3 ± 7.6% 8.3 ± 7.6% 24 6.7 ± 2.4% 10.0 ±4.7%  48 4.2 ± 3.5% 12.5 ± 3.5%  72 0.0 ± 0.0% 0.0 ± 0.0% 120 1.7 ± 0.0%0.8 ± 1.2%

TABLE 6 % Lysis in samples from Bioreactor # 2430 24 hours aftertransfer (±1SD) Time Untreated H₂O₂ (h) Control Treatment 0 0.0 ± 0.0%0.0 ± 0.0% 24 85.8 ± 3.5%  7.5 ± 1.2% 48 75.8 ± 20.0% 0.0 ± 0.0% 96 13.3± 4.7%  4.1 ± 1.2%

TABLE 7 % Lysis in samples from Bioreactor # 2430 48 hours aftertransfer Time Untreated H₂O₂ (h) Control Treatment 0  3.3 ± 2.9%  3.3 ±2.9% 24 63.3 ± 2.4% 25.0 ± 9.4% 96 97.5 ± 1.2% 16.7 ± 4.7%

As shown in Table 5, lysis remained at low levels in both the controland hydrogen peroxide treatment cultures. The results in Tables 6 and 7show lysis occurred at high levels (greater than 80%) in the controlcultures and was held to lower levels with the hydrogen peroxidetreatments (less than 30%), demonstrating a treatment with hydrogenperoxide may successfully reduce lysis by 60-80%.

The findings from the flask test samples were then applied to commercialscale cultures of Haematococcus pluvialis (Strain 1) containing greenswimmer cells that were transferred from open raceway pond bioreactor#2310 disposed in a greenhouse, using paddlewheel mixing, and operatingin conditions: reactor volume of 55,000 L; Daily PAR of 42 mol m⁻² d⁻¹;pH of 7.5; and paddlewheel speed of 40%; to open raceway pond bioreactor#2420 disposed in a greenhouse, using paddlewheel mixing, operating inconditions: reactor volume of 140,000 L; Daily PAR of 40 mol m⁻² d⁻¹; pHof 7.3; and paddlewheel speed of 70%. Similarly, the culture inbioreactor #2320, operating in conditions: reactor volume of 60,000 L;Daily PAR of 42 mol m⁻² d⁻¹; pH of 7.5; and paddlewheel speed of 40%;was transferred by being split into pond bioreactors #2410 and 2430operating in conditions: reactor volume of 150,000 L; Daily PAR of 40mol m⁻² d⁻¹; pH of 7.3; and paddlewheel speed of 70%. The cultures inpond bioreactors #2310 and 2320 had previously received treatments of0.02 mL/L hydrogen peroxide 35% stock concentration (calculatedconcentration of 0.007 mL/L) every six hours, which continued after thecultures were transferred until the fourth day of treatment. Lysis wasquantified upon harvest of the culture through visual observation ofsamples under a microscope. Carotenoid content of the cells was measuredby UV method upon harvest, and the average growth rate of the culturewas calculated based on periodic dry weight samples. The results werecompared to data from previous culture runs in the same bioreactors orbioreactors with similar operating conditions during the same month(September 2014) at the same location. The results are shown in Tables8-10.

TABLE 8 H₂O₂ Maximum % Bioreactor Treatment Lysis of Culture 2410 No 25%2420 No 25% 2430 No 90% 2440 No 95% 2410 Yes 70% 2420 Yes 20% 2430 Yes35%

TABLE 9 H₂O₂ Maximum % Bioreactor Treatment Carotenoid (UV) 2410 No 2.842420 No 0.79 2430 No 1.88 2440 No 2.38 2410 Yes 3.22 2420 Yes 3.58 2430Yes 1.21 [Ended early]

TABLE 10 Average H₂O₂ Growth Rate Bioreactor Treatment (g/m²/day) 2410No 2.1 2420 No 2.3 2430 No 3.2 2440 No 3.6 2410 Yes 4.7 2420 Yes 4.32430 Yes 2.3 [Ended early]

As shown in Table 8, lysis remained in the range of 20-35% in two of thecommercial scale cultures treated with hydrogen peroxide, which was animprovement over the historical data that showed untreated cultures mayreach lysis levels over 80%. As shown in Tables 9 and 10, the culturestreated with hydrogen peroxide produced the higher levels of carotenoidsand had a higher average growth rate than the untreated cultures,indicating that the hydrogen peroxide treatment may be directlyimproving growth and carotenoid accumulation or may be indirectlyimproving growth and carotenoid accumulation by creating an environmentwith suppressed lysis. Viewing the results from the flask tests and thecommercial scale cultures together, treating a culture of Haematococcuspluvialis green swimmer cells with hydrogen peroxide at a calculatedconcentration of at least 0.007 mL/L every 6 hours can reduce the levelof cell lysis by at least 60% without negatively affecting the culturegrowth rate (i.e., biomass accumulation) and accumulation ofcarotenoids.

Example 6

A series of experiments were conducted to evaluate the effectiveness ofhydrogen peroxide alone and in combination with concentrations of saltas a treatment for controlling chytrid infections in Haematococcuspluvialis cultures. Culture samples of Haematococcus pluvialis(Strain 1) cultures were taken and placed into flasks from open racewaybioreactor #2420 disposed within a greenhouse, mixed with paddlewheels,and operating in conditions: reactor volume of 170,000 L; Daily PAR of53 mol m⁻² d⁻¹; pH of 7.3; and paddlewheel speed of 70%. Bioreactor#2420 had just been inoculated with cells in the green swimmer stageinto reddening conditions that comprised of culture medium comprising a1 ppt concentration of sodium chloride (NaCl). The flask experimentconsisted of duplicate untreated controls and duplicate treatments. Thetreated flask cultures were dosed with 0.03 mL/L hydrogen peroxide of25% stock concentration (calculated concentration of 0.0075 mL/L) 2-3times per day. Samples were taken from the flask cultures daily tomonitor the percentage of chytrids in the total cells of the culture (%chytrid infection) through visual observation using a microscope andmeasurement of cell dry weight in g/L for a duration of about 9 days(217 hours). The results are shown in Tables 11-12.

TABLE 11 Average Chytrid Infection (%) Time H₂O₂ (h) Control Treatment 00.0 ± 0.0% 0.0 ± 0.0% 24 0.0 ± 0.0% 0.0 ± 0.0% 48 0.8 ± 1.2% 0.0 ± 0.0%72 0.0 ± 0.0% 0.0 ± 0.0% 144 38.3 ± 54.2% 0.0 ± 0.0% 168 40.0 ± 56.6%0.0 ± 0.0% 197 43.3 ± 44.8% 0.0 ± 0.0% 217 50.0 ± 58.9% 0.0 ± 0.0%

TABLE 12 Average Cell Dry Weight (g/L) Time H₂O₂ (h) Control Treatment 00.1 ± 0.0 0.1 ± 0.0 24 0.1 ± 0.1 0.1 ± 0.0 48 0.3 ± 0.0 0.2 ± 0.0 72 0.5± 0.1 0.3 ± 0.1 144 No data No data 168 0.8 ± 0.2 0.7 ± 0.0 197 0.7 ±0.0 0.6 ± 0.0 217 0.9 ± 0.0 0.8 ± 0.0

Results showed that the % chytrid infection in the untreated controlsvaried widely (15-90%) starting on day six (144 h), with the averagesteadily increasing over the remaining days to over 40% (as shown inTable 11). In the treated cultures, % chytrid infection was 0% theentire nine day period. As shown in Table 12, the dry cell weightincreased in both the control and treated cultures.

Culture samples for a second experiment were taken three days afterinoculation when the salt concentration was at 1 ppt from open racewaybioreactor #2440 disposed within a greenhouse, mixed with paddlewheels,and operating in conditions: reactor volume of 175,000 L; Daily PAR of40 mol m⁻² d⁻¹; pH of 7.3; paddlewheel speed of 70%. Samples were placedin flasks for culturing, consisting of duplicate untreated controls andduplicate treatments. The treated flask cultures were dosed with 0.03mL/L hydrogen peroxide of 25% stock concentration (calculatedconcentration of 0.0075 mL/L) 2-3 times per day. The flask culturescontained Haematococcus pluvialis (Strain 1) cells in both the greenswimmer stage and early red cyst stage. Samples were taken from theflask cultures daily to quantify % chytrid infection and cell dry weightfor a duration of about 6 days (144 hours). The results are shown inTables 13-14.

TABLE 13 Time Average Chytrid Infection (%) (h) Control H₂O₂ Treatment 0 0.0 ± 0.0%  0.0 ± 0.0% 24  0.0 ± 0.0%  1.7 ± 2.9% 96 59.4 ± 7.7% 25.0 ±2.9% 120 68.3 ± 4.4% 51.7 ± 5.0% 144 67.2 ± 4.2% 43.9 ± 4.2%

TABLE 14 Time Average Cell Dry Weight (g/L) (h) Control H₂O₂ Treatment 00.16 ± 0.00 0.16 ± 0.00 24 0.32 ± 0.05 0.31 ± 0.02 96 0.53 ± 0.05 0.55 ±0.04 120 0.56 ± 0.16 0.49 ± 0.09 144 No data No data

As shown in Table 13, the % chytrid infection reached 67% in thecontrols and was reduced in the treated cultures. As shown in Table 14,the dry cell weight increased in both the control and treated cultures.

Culture samples of Haematococcus pluvialis (Strain 1) for a thirdexperiment were collected from open raceway bioreactor #2210 disposedwithin a greenhouse, mixed with a paddlewheel, and operating inconditions: reactor volume of 18,000 L; Daily PAR of 48 mol m⁻² d⁻¹; pHof 7.5; paddlewheel speed of 40%. The collected samples were inoculatedin the green swimmer stage in growth conditions [i.e., in the absence ofsodium chloride (NaCl)]. The samples were distributed in flasks,consisting of duplicate untreated controls and duplicate treatments. Thetreated flask cultures were dosed with 0.03 mL/L hydrogen peroxide of25% stock concentration (calculated concentration of 0.0075 mL/L) twotimes per day. Samples were taken from the flask cultures daily toquantify % chytrid infection and cell dry weight for a duration of about11 days (264 hours). The results are shown in Tables 15-16.

TABLE 15 Time Average Chytrid Infection (%) (h) Control H₂O₂ Treatment 00.0 ± 0.0% 0.0 ± 0.0% 24 0.0 ± 0.0% 0.0 ± 0.0% 48 0.0 ± 0.0% 0.0 ± 0.0%96 0.0 ± 0.0% 0.0 ± 0.0% 120 0.0 ± 0.0% 0.0 ± 0.0% 144 6.7 ± 9.4% 4.2 ±5.9% 168 36.7 ± 37.1% 18.3 ± 25.9% 192 49.2 ± 29.5% 26.6 ± 37.1% 26481.7 ± 2.4%  51.7 ± 7.1% 

TABLE 16 Time Average Cell Dry Weight (g/L) (h) Control H₂O₂ Treatment 00.26 ± 0.00 0.26 ± 0.00 24 0.93 ± 0.04 0.76 ± 0.06 48 No data No data 962.16 ± 0.08 1.90 ± 0.00 120 2.31 ± 0.32 2.26 ± 0.00 144 No data No data168 2.20 ± 0.37  2.2 ± 0.00 192 2.73 ± 0.07 2.68 ± 0.91 264  2.19 ±0.325 2.02 ± 0.65

As shown in Table 15, the % chytrid infection varied widely in the first8 days (192 h), but was reduced in the treatments as compared to thecontrol by day 11 (264 h). As shown in Table 16, the dry cell weightincreased in both the control and treated cultures but was highlyvariable by the end of the experiment.

Culture samples of Haematococcus pluvialis (Strain 1) for a fourthexperiment were collected from open raceway bioreactor #2310 disposedwithin a greenhouse, mixed with a paddlewheel, and operating inconditions: reactor volume of 48,000 L; Daily PAR of 50 mol m⁻² d⁻¹; pHof 7.5; and paddlewheel speed of 40%. The culture samples wereinoculated in the green swimmer stage in growth conditions [i.e., inless than 1 ppt of sodium chloride (NaCl)]. The flask cultures were thendiluted 3:1 with nitrate free HMB media and brought to a 1 pptconcentration of NaCl, consisting of duplicate untreated controls andduplicate treatments. The treated flask cultures were dosed with 0.03mL/L hydrogen peroxide of 25% stock concentration (calculatedconcentration of 0.0075 mL/L) 3 times per day. Samples were taken fromthe flask cultures daily to % chytrid infection and cell dry weight fora duration of about 9 days (216 hours). The results are shown in Tables17-18.

TABLE 17 Time Average Chytrid Infection (%) (h) Control H₂O₂ Treatment 00.0 ± 0.0% 0.0 ± 0.0% 24 0.0 ± 0.0% 0.0 ± 0.0% 48 0.0 ± 0.0% 0.0 ± 0.0%120 16.1 ± 3.5%  1.7 ± 1.7% 144 60.6 ± 7.5%  18.3 ± 18.5% 168 62.8 ±12.5% 17.2 ± 14.4% 192 67.2 ± 5.4%  32.2 ± 28.1% 216 90.6 ± 5.4%  44.4 ±46.8%

TABLE 18 Time Average Cell Dry Weight (g/L) (h) Control H₂O₂ Treatment 00.00 ± 0.00 0.00 ± 0.00 24 0.45 ± 0.03 0.46 ± 0.07 48 0.96 ± 0.15 0.94 ±0.18 120 1.71 ± 0.20 1.60 ± 0.05 144 1.29 ± 0.09 1.56 ± 0.35 168 1.55 ±0.41 2.01 ± 0.02 192 1.21 ± 0.24 2.34 ± 0.39 216 1.77 ± 0.15 2.96 ± 0.26

As shown in Table 17, a 50-90% reduction in % chytrid infection in thetreatments as compared to the control was observed starting 5 days afterinoculation (144 h). The variability in the results were attributed tothe fact that one treatment flask became infected while the other didnot. As shown in Table 18, the dry cell weight increased in both thecontrol and treated cultures but was higher in treated flasks.

Culture samples of Haematococcus pluvialis (Strain 1) were collected fora fifth experiment from open raceway bioreactor #2310 operating inconditions: reactor volume of 66,000 L; Daily PAR of 48 mol m⁻² d⁻¹; pHof 7.5; and paddlewheel speed of 40%. The culture samples weredistributed into flasks for culturing, consisting of duplicate untreatedcontrols and duplicate treatments. The flask cultures were then diluted1:3 into reddening media comprising 1 ppt NaCl or 2 ppt NaCl (33.3 mLand 66.6 mL culture media). The treated flask cultures were dosed with0.03 mL/L hydrogen peroxide of 25% stock concentration (calculatedconcentration of 0.0075 mL/L) 3 times per day. Samples were taken fromthe flask cultures daily to analyze % chytrid infection and cell dryweight for a duration of about 8 days (192 hours). The results are shownin Tables 19-20.

TABLE 19 Average Chytrid Infection (%) 1 ppt 2 ppt 1 ppt NaCl and 2 pptNaCl and Time NaCl H₂O₂ NaCl H₂O₂ (h) Control Treatment ControlTreatment 0 0.0 ± 0.0% 0.0 ± 0.0%  0.0 ± 0.0% 0.0 ± 0.0% 24 0.0 ± 0.0%0.0 ± 0.0%  0.0 ± 0.0% 0.0 ± 0.0% 120 27.5 ± 20.0% 0.0 ± 0.0% 86.7 ±2.4% 0.0 ± 0.0% 144 76.7 ± 9.4%  0.0 ± 0.0% 94.2 ± 5.9% 0.0 ± 0.0% 16893.30 ± 7.1%  0.0 ± 0.0% 100.0 ± 0.0%  0.0 ± 0.0% 192 97.5 ± 1.2%  0.0 ±0.0% 97.5 ± 1.2% 0.0 ± 0.0%

TABLE 20 Average Cell Dry Weight (g/L) 1 ppt 2 ppt 1 ppt NaCl and 2 pptNaCl and Time NaCl H₂O₂ NaCl H₂O₂ (h) Control Treatment ControlTreatment 0 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 24 1.16 ±0.06 1.01 ± 0.04 1.12 ± 0.03 1.00 ± 0.06 120 1.33 ± 0.18 0.89 ± 0.011.12 ± 0.23 0.86 ± 0.06 144 0.95 ± 0.01 0.72 ± 0.06 0.58 ± 0.14 0.66 ±0.08 168 0.64 ± 0.06 0.78 ± 0.03 0.60 ± 0.03 0.78 ± 0.03 192 0.91 ± 0.071.01 ± 0.04 0.66 ± 0.03 1.00 ± 0.03

As shown in Table 19, a chytrid infection appeared within 5 days (120 h)and increased to greater than 90% chytrid infection by day 7 (168 h) inthe control cultures. In the treated cultures, % chytrid infection was0% the entire eight day period. As shown in Table 20, the dry cellweight was comparable between control and treated cultures. Across allexperiments, treatment with hydrogen peroxide was show to be effectivein reducing chytrid infections when used with and without salt.

Example 7

An experiment was conducted to evaluate the effectiveness of thecombination of salt and hydrogen peroxide as a treatment method ofreducing chytrids in an infected culture of Haematococcus pluvialis.Culture samples of Haematococcus pluvialis (Strain 2) were collectedfrom 3,000 L open raceway pond bioreactor #MP1 with paddlewheel mixingdisposed outdoors. At the time the samples was taken, the culturecomprised green swimmer cells and chytrids (100% of culture infected).One mL of infected culture was inoculated into replicate flasks (100 mLvolume) containing axenic green swimmers from a carboy bioreactorculture diluted into reddening media 1 part culture to 3 parts mediacomprising either 1 or 3 ppt salt (NaCl). The flask cultures containing1 ppt salt were dosed three times per day (morning, noon and evening)with 0.02 or 0.04 mL/L of hydrogen peroxide 35% stock concentration(calculated concentrations of 0.007 or 0.014 mL/L). The flask culturescontaining 3 ppt salt were dosed three times per day (morning, noon andevening) with 0.02 mL/L of hydrogen peroxide 35% stock concentration(calculated concentration of 0.007 mL/L). The percent of the cellsinfected with chytrids was quantified via visual observation under amicroscope at 72 and 96 hours after the cultures were inoculated in theflask. The results are shown in Table 21.

TABLE 21 % Chytrids Infection 1 ppt salt 1 ppt salt 3 ppt salt TimeUntreated & 0.007 & 0.0014 & 0.007 (h) Control mL/L H₂O₂ mL/L H₂O₂ mL/LH₂O₂ 72 71.7 ± 0.0% 57.5 ± 5.9% 36.7 ± 4.7% 72.5 ± 1.2% 96 96.7 ± 4.7%NO DATA 97.5 ± 1.2% NO DATA

As shown in Table 21, the hydrogen peroxide treatments for the culturescontaining 1 ppt salt showed a reduction in the chytrids infection %compared to the untreated control and treatment containing 3 ppt saltafter 72 hours, with the 0.014 mL/L hydrogen peroxide treatmentresulting in a larger reduction than the 0.007 mL/L hydrogen peroxidetreatment. After 96 hours the 1 ppt salt and 0.014 mL/L hydrogenperoxide treatment did not appear to continue as an effective treatmentfor the reduction of chytrids, demonstrating that the combination ofsalt and hydrogen peroxide may be effective for reducing chytrids in aculture of Haematococcus with an existing infection for a limited periodof time after which additional action may need to be taken in the formof harvesting the Haematococcus cells or utilizing a different treatmentmethod.

Example 8

An experiment was conducted to evaluate the effectiveness of thecombination of salt and hydrogen peroxide as a treatment method forreducing chytrids in an infected culture of Haematococcus pluvialis.Three 3,000 L open raceway pond bioreactor #'s MP1, MP2, & MP3 withpaddlewheel mixing disposed outdoors were inoculated with cultures ofgreen swimmer Haematococcus pluvialis (Strain 2) cells from outdoorreactor #2310 at a dilution ratio of 1 part culture samples to 3 partsreddening media containing 1 ppt salt (NaCl). After the cultures failedto become infected on their own, chytrid infection was promoted byadding 5 L of infected culture from another outdoor reactor (#2420)after 6 days. MP1 and MP3 received treatments of 0.03 mL/L of hydrogenperoxide 35% stock concentration (calculated concentration of 0.0105mL/L) every 6 hours. MP2 was not treated with hydrogen peroxide to serveas an untreated control for comparison purposes. The percent of cellsinfected with chytrids was quantified using visual observation under amicroscope 24 hours after the chytrids were introduced, and determinedto be 20% in MP1, 10% in MP2, and less than 5% in MP3. After hydrogenperoxide treatments began, the percent of the cultures infected withchytrids, percent of carotenoids by UV method, and cell dry weight (g/L)were quantified daily. The results are show in Tables 22-24.

TABLE 22 % Chytrids Infection 0.0105 mL/L Untreated 0.0105 mL/L H₂O₂(MP3, Time Control (MP2, H₂O₂ (MP1, initial (days after initial initialinfection inoculation) infection 10%) infection 20%) less than 5%) 5 NODATA NO DATA NO DATA 6 NO DATA NO DATA NO DATA 7 8.3 ± 7.6% 23.3 ± 5.8% 1.7 ± 2.9% 8 10.0 ± 5.0%  20.0 ± 5.0%  0.0 ± 0.0% 9 16.7 ± 12.6% 30.0 ±10.0% 1.7 ± 2.9% 10 45.0 ± 18.0% 26.7 ± 2.9%  0.0 ± 0.0% 11 76.7 ± 5.8% 61.7 ± 24.7% 0.0 ± 0.0% 12 90.0 ± 10.0% 63.3 ± 16.1% 0.0 ± 0.0% 13 95.0± 5.0%  73.3 ± 7.6%  3.3 ± 2.9% 14 98.3 ± 2.9%  76.7 ± 17.6% 0.0 ± 0.0%

TABLE 23 % Carotenoids (UV) 0.0105 mL/L Untreated 0.0105 mL/L H₂O₂ (MP3,Time Control (MP2, H₂O₂ (MP1, initial (days after initial initialinfection inoculation) infection 10%) infection 20%) less than 5%) 5 NODATA NO DATA NO DATA 6 NO DATA NO DATA NO DATA 7 NO DATA NO DATA NO DATA8 3.72 3.71 3.77 9 3.72 4.03 4.19 10 4.20 4.44 4.55 11 3.24 4.33 3.74 123.27 3.95 4.47 13 NO DATA NO DATA NO DATA 14 NO DATA NO DATA NO DATA

TABLE 24 Cell Dry Weight (g/L) 0.0105 mL/L Untreated 0.0105 mL/L H₂O₂(MP3, Time Control (MP2, H₂O₂ (MP1, initial (days after initial initialinfection inoculation) infection 10%) infection 20%) less than 5%) 50.156 0.216 0.190 6 0.219 0.283 0.201 7 0.244 0.321 0.311 8 0.314 0.3710.412 9 0.317 0.349 0.353 10 0.346 0.370 0.343 11 0.347 0.353 0.349 120.375 0.357 0.375 13 0.366 0.364 0.390 14 0.360 0.357 0.427

As shown in Table 22, the hydrogen peroxide and salt treatment waseffective for maintaining the chytrids infection level below 5% in theMP3 culture which was below 5% when the treatments started. The hydrogenperoxide and salt treatment was also effective in slowing the chytridinfection of the MP1 culture which started with a moderate level (20%)as compared to the untreated control (MP2). Visual observation of thered cyst cells under a microscope also showed that the Haematococcuscells were susceptible to infection when the cells were transitioningfrom the green swimmer stage to the red cyst stage before the cyst hadfully formed.

As shown in Tables 23 and 24, the continuous treatments with hydrogenperoxide in the presence of salt did not inhibit the accumulation ofcarotenoids and biomass in the Haematococcus cells. Together theseresults demonstrate that continuously treating a culture ofHaematococcus cells infected by chytrids with hydrogen peroxide in thepresence of low salt (1 ppt) is effective in preventing an increase inthe infection if treatment is started when the infection is at a lowlevel (below 5%), or at a minimum the treatment is effective in slowingdown the increase in the infection that is already at a moderate level(e.g., 20%) at the time treatment is initiated without negativelyaffecting the carotenoid (e.g., astaxanthin) and biomass accumulation byHaematococcus.

Example 9

An experiment was conducted to evaluate if higher concentrations[greater than 0.03 mL/L 35% stock (calculated concentrations greaterthan 0.0105 mL/L)] of hydrogen peroxide could be used as a treatmentmethod for reducing chytrids in an infected culture of Haematococcuspluvialis. Culture samples of Haematococcus pluvialis (Strain 2) werecollected on day 5 of the culture from open raceway bioreactor #2420operating in conditions: reactor volume of 170,000 L; Daily PAR notmeasured; pH of 7.3; and paddlewheel speed of 70%). Culture samples weredistributed into flasks for culturing, consisting of duplicate untreatedcontrols and duplicate treatments. Treatments compared differentfrequency doses (one to three times daily) of higher concentrations(0.04 and 0.05 mL/L) of hydrogen peroxide 35% stock concentration(calculated concentrations of 0.0140 and 0.0175 mL/L) with the standardtreatment to date (calculated concentration of 0.0105 mL/L three timesdaily). Samples were taken from the flask cultures every few days inorder to quantify % lysis, % chytrid infection, and cell dry weight fora duration of about 11 days. Due to lysis rates exceeding 10% in alltreatments by day 5, treatment was discontinued and recovery from lysiswas monitored. Results are shown in Table 25-27.

TABLE 25 % Lysis 0.0105 mL/L 0.0140 mL/L 0.0140 mL/L 0.0140 mL/L 0.0175mL/L Time (days H₂O₂ H₂O₂ 1 H₂O₂ 2 H₂O₂ 3 H₂O₂ 1 after Untreated 3 timesper time per times per times per time per inoculation) Control day dayday day day 0 4.2 ± 1.2 4.2 ± 1.2 4.2 ± 1.2 4.2 ± 1.2 4.2 ± 1.2 4.2 ±1.2 3 0.8 ± 1.2 0.8 ± 1.2 5.0 ± 2.4 5.0 ± 4.7 2.5 ± 3.5 2.5 ± 1.2 4 4.2± 5.9 3.3 ± 2.4 4.2 ± 1.2 3.3 ± 2.4 9.2 ± 1.2 4.2 ± 1.2 5 1.7 ± 0.0 10.8± 1.2  10.0 ± 2.4  10.0 ± 2.4  42.5 ± 15.3 9.2 ± 1.2 6 0.0 ± 0.0 3.3 ±2.4 7.5 ± 1.2 9.7 ± 5.9 55.0 ± 30.6 6.7 ± 2.4 9 No Data 6.7 ± 2.4 10.8 ±1.2  5.0 ± 0.0 60.0 ± 28.3 6.7 ± 4.7 11 No Data 4.2 ± 3.5 7.5 ± 1.2 0.0± 0.0 56.7 ± 33.0 4.2 ± 3.5

TABLE 26 % Chytrid infection 0.0105 mL/L 0.0140 mL/L 0.0140 mL/L 0.0140mL/L 0.0175 mL/L Time (days H₂O₂ H₂O₂ 1 H₂O₂ 2 H₂O₂ 3 H₂O₂ 1 afterUntreated 3 times per time per times per times per time per inoculation)Control day day day day day 0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.00.0 ± 0.0 0.0 ± 0.0 3 28.3 ± 23.6 0.8 ± 1.2 0.0 ± 0.0 2.5 ± 1.2 3.3 ±4.7 0.8 ± 1.2 4 41.7 ± 18.8 2.5 ± 3.5 0.0 ± 0.0 2.5 ± 3.5 0.0 ± 0.0 1.7± 0.0 5 53.3 ± 0.0  1.7 ± 0.0 1.7 ± 2.4 5.0 ± 4.7 0.0 ± 0.0 1.7 ± 0.0 688.3 ± 2.4  2.5 ± 1.2 2.5 ± 1.2 5.8 ± 3.5 0.0 ± 0.0 0.0 ± 0.0 9 97.5 ±3.5  7.5 ± 3.5 0.0 ± 0.0 43.3 ± 4.7  0.0 ± 0.0 0.0 ± 0.0 11 No Data100.0 ± 0.0  35.8 ± 29.5 97.5 ± 3.5  11.7 ± 16.5 34.2 ± 27.1

TABLE 27 Culture Dry Weight (g/L) 0.0105 mL/L 0.0140 mL/L 0.0140 mL/L0.0140 mL/L 0.0175 mL/L Time (days H₂O₂ H₂O₂ 1 H₂O₂ 2 H₂O₂ 3 H₂O₂ 1after Untreated 3 times per time per times per times per time perinoculation) Control day day day day day 0 0.29 ± 0.02 0.29 ± 0.02 0.29± 0.02 0.29 ± 0.02 0.29 ± 0.02 0.29 ± 0.02 3 0.42 ± 0.07 0.42 ± 0.070.49 ± 0.05 0.44 ± 0.0  0.40 ± 0.01 0.47 ± 0.03 4 No Data No Data NoData No Data No Data No Data 5 No Data No Data No Data No Data No DataNo Data 6 No Data 0.45 ± 0.02 0.44 ± 0.0  0.41 ± 0.11 0.30 ± 0.06 0.35 ±0.02 9 0.59 ± 0.29 0.74 ± 0.02 0.82 ± 0.04 0.67 ± 0.01 0.54 ± 0.11 0.76± 0.01 11 0.57 ± 0.16 0.69 ± 0.09 0.54 ± 0.32 0.64 ± 0.08 0.81 ± 0.1 0.79 ± 0.13

As shown in Table 25, percent lysis reached 10% in all cultures treatedwith hydrogen peroxide by day 5, except for 0.014 mL/L dosed three timesper day which were at 40% lysis. Treatment was discontinued at this timeand the lysis rate remained stable through days 6 to 11. As shown inTable 26, chytrid infection increased to 100% in the untreated controls,but remained less than 10% in all treated flasks until 4-6 days aftertreatment ended. Final infection rates were lowest in flasks previouslydosed with 0.0140 and 0.0175 mL/L once per day. As shown in Table 27,culture dry weights were highest in the cultures treated once daily onday 9. While the results showed that the treatments did not demonstratea negative effect on lysis, the treatments were effective againstchytrid infection without negatively affecting dry weight.

Example 10

A second flask experiment was conducted to evaluate if one dailytreatment of 0.03-0.05 mL/L 35% stock concentration of hydrogen peroxide(calculated concentration 0.0105-0.0175 mL/L) could be used as atreatment method for reducing chytrids in an infected culture ofHaematococcus pluvialis. Culture samples of Haematococcus pluvialis(Strain 2) were collected on the second day of the culture from openraceway bioreactor #2430 operating in conditions: reactor volume of170,000 L; Daily PAR not measured; pH of 7.3; and paddlewheel speed of70. The culture samples were distributed into flasks for culturing,consisting of duplicate untreated controls and duplicate treatments.Treatments compared different frequency doses (once daily and once dailyfollowed by every other day) of 0.03, 0.04 and 0.05 mL/L 35% stockhydrogen peroxide (calculated concentrations of 0.0105, 0.0140, and0.0175 mL/L). Samples were taken from the flask cultures daily toquantify % lysis and % chytrid infection and cell dry weight wasquantified every other day for a duration of 11 days. The results areshown in Tables 28-30.

TABLE 28 % Lysis 0.0175 mL/L 0.0105 mL/L 0.0140 mL/L H₂O₂ 1 H₂O₂ 1 H₂O₂1 time per 0.0105 mL/L time per 0.0140 mL/L time per day for 2 d Time(days H₂O₂ day for 3 d H₂O₂ 1 day for 3 d then after Untreated 1 timeper then every time per then every every inoculation) Control day otherday day other day other 0 0.8 ± 2.0 0.8 ± 2.0 0.8 ± 2.0 0.8 ± 2.0 0.8 ±2.0 0.8 ± 2.0 3 1.7 ± 2.4 0.0 ± 0.0 0.0 ± 0.0 4.2 ± 1.2 4.2 ± 1.2 31.7 ±11.8 4 0.0 ± 0.0 1.7 ± 0.0 1.7 ± 0.0 10.0 ± 8.7  10.0 ± 8.7  22.2 ± 8.5 5 0.0 ± 0.0 1.7 ± 0.0 1.7 ± 2.4 10.0 ± 2.4  15.8 ± 5.9  19.2 ± 17.6 60.0 ± 0.0 0.8 ± 2.0 3.3 ± 4.7 9.2 ± 3.5 6.7 ± 0.0  25 ± 0.0 7 1.7 ± 0.00.0 ± 0.0 0.0 ± 0.0 20.0 ± 9.4  13.3 ± 4.7  No Data 8 5.8 ± 1.2 1.7 ±2.4 0.8 ± 1.2 6.7 ± 7.0 13.3 ± 2.4  No Data 9 0.8 ± 1.2 7.5 ± 8.3 0.8 ±1.2 21.7 ± 7.1  5.0 ± 2.4 No Data 10 6.7 ± 2.4 0.0 ± 0.0 0.8 ± 1.2 9.2 ±3.5 15.8 ± 12.9 No Data

TABLE 29 % Chytrid infection 0.0175 mL/L 0.0105 mL/L 0.0140 mL/L H₂O₂ 1H₂O₂ 1 H₂O₂ 1 time per 0.0105 mL/L time per 0.0140 mL/L time per day for2 d Time (days H₂O₂ day for 3 d H₂O₂ 1 day for 3 d then after Untreated1 time per then every time per then every every inoculation) Control dayother day day other day other 0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.00.0 ± 0.0 0.0 ± 0.0 3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.00.0 ± 0.0 4 14.2 ± 8.3  0.8 ± 1.2 0.8 ± 1.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ±0.0 5 21.7 ± 4.7  0.8 ± 1.2 5.0 ± 2.4 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 636.7 ± 16.5 1.7 ± 2.4 2.5 ± 1.2 0.0 ± 0.0 1.7 ± 2.4 0.8 ± 1.2 7 17.5 ±3.5  3.3 ± 0.0 0.0 ± 0.0 1.7 ± 2.4 0.0 ± 0.0 No data 8 20.0 ± 7.1  0.0 ±0.0 0.0 ± 0.0 0.8 ± 1.2 0.8 ± 1.2 No data 9 21.7 ± 7.1  0.8 ± 1.2 2.5 ±3.5 0.8 ± 1.2 0.0 ± 0.0 No data 10 26.7 ± 14.1 0.0 ± 0.0 1.7 ± 2.4 0.0 ±0.0 0.0 ± 0.0 No data

TABLE 30 Culture Dry Weight (g/L) 0.0175 mL/L 0.0105 mL/L 0.0140 mL/LH₂O₂ 1 H₂O₂ 1 H₂O₂ 1 time per 0.0105 mL/L time per 0.0140 mL/L time perday for 2 d Time (days H₂O₂ day for 3 d H₂O₂ 1 day for 3 d then afterUntreated 1 time per then every time per then every every inoculation)Control day other day day other day other day 0 0.05 ± 0.01 0.05 ± 0.010.05 ± 0.01 0.05 ± 0.01 0.05 ± 0.01 0.05 ± 0.01 3 No data No data Nodata No data No data No data 4 0.35 ± 0.03 0.32 ± 0.01 0.32 ± 0.01 0.32± 0.01 0.32 ± 0.01 0.25 ± 0.02 5 No data No data No data No data No dataNo data 6 0.37 ± 0.02 0.35 ± 0.08 0.29 ± 0.01 0.30 ± 0.03 0.35 ± 0.010.24 ± 0.03 7 No Data No Data No Data No Data No Data No Data 8 0.54 ±0.06 0.42 ± 0.01 0.49 ± 0.04 0.41 ± 0.05 0.48 ± 0.01 No Data 9 No DataNo Data No Data No Data No Data No Data 10 0.39 ± 0.04 0.38 ± 0.01 0.40± 0.00 0.35 ± 0.04 0.38 ± 0.01 No Data

As shown in Table 28, percent lysis remained under 10% for controlcultures and those treated with 0.0105 mL/L hydrogen peroxide for 11days. Those treated with 0.0140 mL/L hydrogen peroxide reached 20% lysisby day 7 and those treated with 0.0175 mL/L reached 30% lysis by day 3after only being dosed twice. These flask cultures were discarded on day6. Cultures in this set may have been less tolerant to hydrogen peroxidebecause they were pulled back from the parent reactor within two days ofinoculation while in the previous experiment (Example 9), culturesamples were pulled back 5 days after inoculation of the parent and werelikely further along in cyst stage. As shown in Table 29, chytridinfection reached 20-40% in the untreated controls, but remained lessthan 10% in all treated flasks. As shown in Table 30, culture dryweights were slightly reduced in flasks treated every day compared tocontrols and flasks treated every day for 3 days and then every otherday. The results show that lower concentrations of hydrogen peroxide arecapable of reducing both lysis and chytrid infections, while the highconcentrations of hydrogen peroxide were more effective for reducingchytrid infections than lysis.

Example 11

A second flask experiment was conducted to fine tune treatment of 0.03mL/L 35% stock concentration of hydrogen peroxide (calculatedconcentration of 0.0105 mL/L) for reducing chytrids in an infectedculture of Haematococcus pluvialis. Culture samples of Haematococcuspluvialis (Strain 2) were collected on the second day of culture fromopen raceway bioreactor #2410 operating in conditions: reactor volume of150,000 L; Daily PAR of 9.77 mol m⁻² d⁻¹; pH of 7.3; and paddlewheelspeed of 70%. Culture samples were distributed into flasks forculturing, consisting of duplicate untreated controls and duplicatetreatments. Treatments compared different frequency doses (once daily,once every other day and once daily for 3 days only) of 0.03 mL/L of 35%stock hydrogen peroxide (calculated concentration of 0.0105 mL/L).Samples were taken from the flask cultures daily to quantify % lysis and% chytrid infection and cell dry weight was quantified every other dayfor a duration of 11 days. The results are shown in Tables 31-33.

TABLE 31 % Lysis 0.0105 mL/L 0.0105 mL/L Time 0.0105 mL/L H₂O₂ 1 timeH₂O₂ 1 time (days after Untreated H₂O₂ 1 time every other daily forinoculation) Control per day day 3 days 1 5.0 ± 5.0 5.0 ± 5.0 5.0 ± 5.05.0 ± 5.0 2 8.3 ± 0.0 2.5 ± 1.2 3.3 ± 2.4 3.3 ± 0.0 3 3.3 ± 2.4 5.8 ±3.5 4.2 ± 1.2 7.5 ± 1.2 4 3.3 ± 0.0 4.2 ± 1.2 3.3 ± 0.0 0.8 ± 1.2 5 1.7± 2.4 4.2 ± 3.5 3.3 ± 2.4 5.0 ± 0.0 6 0.0 ± 0.0 0.0 ± 0.0 3.3 ± 2.4 4.2± 1.2 7 0.0 ± 2.4 2.4 ± 0.0 3.3 ± 0.0 1.7 ± 2.4 8 No data No data Nodata No data

TABLE 32 % Chytrid Infection 0.0105 mL/L 0.0105 mL/L Time 0.0105 mL/LH₂O₂ 1 time H₂O₂ 1 time (days after Untreated H₂O₂ 1 time every otherdaily for inoculation) Control per day day 3 days 1 0.0 ± 0.0 0.0 ± 0.00.0 ± 0.0 0.0 ± 0.0 2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 3 2.5 ±3.5 0.0 ± 0.0 2.5 ± 1.2 0.0 ± 0.0 4 1.7 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ±0.0 5 23.3 ± 4.7  2.5 ± 3.5 0.8 ± 1.2 0.8 ± 1.2 6 48.3 ± 4.7  0.0 ± 0.03.3 ± 0.0 0.0 ± 0.0 7 92.5 ± 3.5  0.0 ± 0.0 0.8 ± 1.2 1.7 ± 2.4 8 Nodata No data No data No data

TABLE 33 Culture dry weight (g/L) 0.0105 mL/L 0.0105 mL/L Time 0.0105mL/L H₂O₂ 1 time H₂O₂ 1 time (days after Untreated H₂O₂ 1 time everyother daily for inoculation) Control per day day 3 days 1 0.066 ± 0.0 0.066 ± 0.0  0.066 ± 0.0  0.066 ± 0.0  2 0.255 ± 0.021 0.210 ± 0.0280.215 ± 0.007 No data 4 0.355 ± 0.049 0.308 ± 0.011 0.331 ± 0.011 0.285± 0.0  6 0.500 ± 0.071 0.395 ± 0.007 0.455 ± 0.021 0.475 ± 0.035 7 0.475± 0.007 0.440 ± 0.028 0.540 ± 0.042 0.530 ± 0.014 8 0.360 ± 0.057 0.490± 0.014 0.510 ± 0.014 0.560 ± 0.028

As shown in Table 31, cell lysis remained below 10% in flasks acrosstreatments. As shown in Table 32, chytrid infection remained below 5% inall treated flasks but increased in the untreated control after day 4 togreater than 90% infection. As shown in Table 33, culture dry weightincreased in every treatment but noticeably decreased in infectedcontrols after day 6. These results suggest that a conservativetreatment of 0.0105 mL/L hydrogen peroxide applied every day, everyother day or every day for 3 days only would be sufficient forpreventing chytrids in reddening cultures of Haematococcus pluvialiswhile also not affect biomass accumulation.

Example 12

An experiment was conducted to evaluate the effectiveness of salt as atreatment method for reducing chytrids in an infected culture ofHaematococcus pluvialis, without the addition of hydrogen peroxide. Thistest was done side by side in flasks (0.1 L) and in reactors located ina greenhouse operating in conditions: reactor volume of 230 L; Daily PARof 20 mol m⁻² d⁻¹; pH (not measured); and paddlewheel speed of 21 RPM.Flasks were inoculated (100 mL volume) with samples from a culture ofgreen swimmer stage Haematococcus pluvialis (Strain 2) cells from openraceway pond outdoor reactor #2410 operating in conditions: reactorvolume of 193,750 L; pH of 7.3; and paddlewheel speed of 70%; andbrought up to concentration of 2 ppt salt (NaCl). The flask cultureswere mixed by shaking at 140 rpm and received 300-400 μmol/m² s ofphotosynthetically active radiation (PAR) in 12 hour light cycles andconstant supply of 2.5% CO₂. The motility of the culture was monitoredby visual observation under a microscope. Once the motility of the cellswas observed to be below 10%, the level of salt was elevated induplicate to 4-11 ppt and compared to the culture in two flasksremaining at 2 ppt salt. Nine 230 L reactors were also seeded withculture from bioreactor #2410 on the same day as the flasks and broughtto 2 ppt concentration of salt. Once motility of the cells was reducedto <10% (day 3 of culture for flasks and day 2 for 230 L reactors) thelevel of salt was elevated to a point between 4-18 ppt and compared tothe culture in two reactors remaining at 2 ppt salt. Flasks and 230 Lreactors were monitored for percentage of cell lysis via visualobservation under a microscope, culture dry weight (biomass accumulationin g/L), and percentage of chytrid infection via visual observationunder a microscope. Carotenoid production, a proxy for determiningastaxanthin, was quantified every other day of culture in the 230 Lreactors using the UV method. Results are shown in Tables 34-40.

TABLE 34 % Cell Lysis in Flasks NaCl concentration (ppt) Time (d) 2 4 56 7 8 9 10 11 9 0 ± 0% 0 ± 0% 0 ± 0% 4 ± 3% 0 ± 0% 3 ± 2% 0 ± 0% 1 ± 1%0 ± 0% 11 0 ± 0% 0 ± 0% 1 ± 1% 5 ± 4% 7 ± 3% 2 ± 0% 2 ± 0% 3 ± 1% 1 ± 1%

TABLE 35 % Cell Lysis in 230L reactors NaCl concentration (ppt) Time (d)2 2 4 5 6 7 8 10 18 2 0.00% 0.00% 1.67% 3.18% 0.00% 0.00% 3.18% 0.00%0.00% 3 1.67% 0.00% 0.00% 5.00% 0.00% 0.00% 3.33% 5.00% 3.33% 4 0.00%1.67% 0.00% 3.33% 8.33% 3.33% 0.00% 6.67% 13.33% 5 0.00% 0.00% 3.33%1.67% 0.00% 1.67% 3.33% 36.67% 5.00% 6 0.00% 0.00% 0.00% 21.67% 13.33%18.33% 16.67% 31.67% 26.67% 7 0.00% 3.33% 30.00% 10.00% 18.33% 8.33%5.00% 3.33% 20.00% 8 0.00% 1.67% 23.33% 1.67% 1.67% 1.67% 0.00% 0.00%1.67% 9 0.00% 30.00% 13.33% 5.00% 6.67% 6.67% 6.67% 5.00% 8.33% 10 0.00%0.00% 11.67% 5.00% 1.67% 6.67% 15.00% 3.33% 5.00% 11 1.67% no data 5.00%8.33% 6.67% 11.67% 11.67% 6.67% 1.67% 12 0.00% no data 6.67% 3.33% 5.00%5.00% 3.33% 5.00% 3.33%

TABLE 36 % Chytrid infection in Flasks NaCl concentration (ppt) Time (d)2 4 5 6 7 8 9 10 11 9 82 ± 17% 22 ± 20% 18 ± 16%  8 ± 11% 3 ± 5% 1 ± 1%1 ± 1% 0 ± 0% 0 ± 0% 11 96 ± 4%  85 ± 16% 53 ± 9%  19 ± 27% 0 ± 0% 2 ±2% 1 ± 1% 0 ± 0% 17 ± 24%

TABLE 37 % Chytrid infection in 230L reactors NaCl concentration (ppt)Time (d) 2 2 4 5 6 7 8 10 18 2 1.67% 0.00% 0.00% 1.67% 1.67% 3.33% 0.00%0.00% 0.00% 3 0.00% 0.00% 1.67% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 40.00% 0.00% 0.00% 1.67% 1.67% 0.00% 0.00% 1.67% 0.00% 5 0.00% 0.00%0.00% 0.00% 1.67% 1.67% 1.67% 0.00% 3.33% 6 0.00% 0.00% 0.00% 0.00%1.67% 0.00% 0.00% 0.00% 0.00% 7 8.33% 3.33% 1.67% 8.33% 5.00% 5.00%0.00% 0.00% 0.00% 8 16.67% 21.67% 0.00% 0.00% 3.33% 1.67% 0.00% 0.00%1.67% 9 45.00% 10.00% 11.67% 10.00% 0.00% 3.33% 0.00% 1.67% 3.33% 1071.67% 100.00% 30.00% 3.33% 1.67% 5.00% 1.67% 0.00% 0.00% 11 81.67% nodata 36.67% 13.33% 0.00% 0.00% 1.67% 6.67% 1.67% 12 100.00% no data65.00% 28.33% 15.00% 3.33% 3.33% 3.33% 1.67%

TABLE 38 Culture Dry Weight (g/L) in Flasks Time NaCl concentration(ppt) (d) 2 4 5 6 7 8 9 10 11 0 0.044 0.044 0.044 0.044 0.044 0.0440.044 0.044 0.044 11 0.49 ± 0.04 0.48 ± 0.05 0.43 ± 0.04 0.48 ± 0.020.49 ± 0.02 0.50 ± 0.04 0.45 ± 0.00 0.42 ± 0.01 0.40 ± 0.04

TABLE 39 Culture Dry Weight (g/L) in 230L reactors Time NaClconcentration (ppt) (d) 2 2 4 5 6 7 8 10 18 0 0.030 0.043 0.037 0.0400.040 0.035 0.018 0.045 0.035 1 0.042 0.048 0.063 0.070 0.058 0.0480.057 0.060 0.052 2 0.065 0.072 0.080 0.095 0.085 0.103 0.093 0.0880.098 3 0.120 0.105 0.103 0.143 0.133 0.123 0.135 0.140 0.158 4 0.1450.113 0.115 0.162 0.140 0.145 0.145 0.120 0.135 5 0.228 0.125 0.1200.185 0.178 0.150 0.168 0.130 0.173 6 0.173 0.130 0.125 0.213 0.1980.170 0.193 0.168 0.180 7 0.185 0.150 0.148 0.223 0.218 0.198 0.2050.145 0.165 8 0.273 0.170 0.153 0.270 0.260 0.260 0.237 0.145 0.165 90.145 0.218 0.198 0.293 0.260 0.148 0.218 0.160 0.175 10 0.220 0.1500.143 0.265 0.250 0.205 0.257 0.173 0.183 11 0.145 no data 0.148 0.2500.282 0.218 0.220 0.145 0.200 12 0.187 no data 0.150 0.280 0.323 0.2450.263 0.168 0.175

TABLE 40 % Carotenoid by UV Method in 230L reactors Time NaClconcentration (ppt) (d) 2 2 4 5 6 7 8 10 18 3 1.38 1.32 1.3 1.26 1.711.76 1.25 1.48 1.34 5 2.53 2.14 2.6 3.12 2.67 2.82 2.02 1.96 1.81 7 3.773.69 3.87 5.12 4.57 4.28 4.11 2.82 2.63 9 3.41 3.93 3.56 5.33 4.77 4.734.83 3.43 2.79 11 3.28 no data 5.02 5.45 5.67 5.3 5.41 6.11 4.25 12 3.16no data 3.07 5.74 5.63 5.72 5.71 4.38 3.79

As shown in Tables 34 and 35, cell lysis remained below 10% in flasksfor all treatments, and below 30% in 230 L reactors across alltreatments except 10 ppt. Lysis was highest at 4, 10 and 18 ppt salt. Asshown in Table 36 and 37, chytrid infection decreased as salinityincreased for both flasks and 230 L reactors. Chytrid infection remainedbelow 5-10% for treatments between 7-10 ppt in flasks, and treatments at7 ppt and above in 230 L reactors. As shown in Tables 38, the biomassaccumulation measured by culture dry weight was similar for alltreatments except the two highest salinities, which were reduced. Asshown in Table 39, biomass accumulation was highest in the 230 Lreactors after day 7 for treatments between 5-8 ppt. As shown in Table40, carotenoid accumulation was highest for treatments between 5-8 pptin the 230 L reactors. Visual observation under a microscope showed thatcysts in cultures set to 8 ppt were the healthiest and cleanest looking(e.g., large, red, and least fouled) in both flasks and 230 L reactors.These findings suggest that as an alternative to hydrogen peroxidetreatments, 8 ppt salt can be used to reduce chytrid infection whilemaintaining biomass and astaxanthin accumulation.

Example 13

Experiments were performed to determine chytrid tolerance to bleach,salt, Lufenuron (chemical biocide), and Rid Fungus (blend of naturalorganic herbs) for evaluation as a treatment for chytrids. A purechytrid culture in a well plate was treated every 24 hours and observedunder a microscope to determine the effect. An untreated culture wasused as a control for comparison purposes. Qualitative assessments andquantitative estimates were made by visual observation under amicroscope.

In the control culture, sporangia and zoospores without tails wereobserved after 1 hour. Swimming zoospores were observed after 24 hours,sporangia and swimming zoospores (80-100% motility) were observed after48 hours, and swimming zoospores (90-100%) motility were observed after72 hours. From the observation of the control culture, a treatment thatreduces the formation or motility of zoospores may be furtherinvestigated as a viable treatment.

In the culture receiving 0.03 mL/L of bleach 12.5% stock concentration(calculated concentration of 0.00375 mL/L), sporangia and zoospores wereobserved after 24 hours. Sporangia and swimming zoospores (100%motility) were observed after 48 hours, and swimming zoospores (100%motility) were observed after 72 hours.

In the culture receiving 0.1 mL/L of bleach 12.5% stock concentration(calculated concentration of 0.0125 mL/L), sporangia only was observedafter 1 hour. Only bacteria were observed after 24 hours, bacteria and afew zoospores were observed after 48 hours, and bacteria with nozoospores and a few sporangia were observed after 72 hours.

In the culture receiving 0.2 mL/L of bleach 12.5% stock concentration(calculated concentration of 0.025 mL/L), sporangia and zoospores wereobserved after 1 hour. Swimming zoospores were observed after 24 hours,and bacteria and sporangia only (no zoospores) were observed after 48hours and 72 hours. The lack of zoospores after 72 hours for the 0.0125and 0.025 mL/L bleach treatments indicate that the treatment may beeffective against chytrids, but the known tolerance of Haematococcuspluvialis (Strain 1) is 0.00375 mL/L and thus the higher concentrationswould not be viable as a treatment during culturing.

In the culture receiving 20 ppt salt (NaCl), clumped sporangia andzoospores were observed after 1 hour. Wilting sporangia and no zoosporeswere observed after 24 hours, bacteria with shriveled zoospores andsporangia were observed after 48 hours, and no zoospores or sporangiawere observed after 72 hours. The results show that the high level ofsalt is effective against chytrids, but the level is above the thresholdthat has been shown to negatively affect some strains of Haematococcusduring culturing.

In the culture receiving 40 ppm of Lufenuron, swimming zoospores wereobserved after 1 hour. Swimming zoospores were again observed after 24hours, and heavy bacteria with sporangia and zoospores were observedafter 48 hours and 72 hours. The resulting proliferation of zoosporesdemonstrates that Lufenuron is not effective as a chytrid treatment.

In the culture receiving 0.01% of Rid Fungus, swimming zoospores wereobserved after 24 hours. Bacteria only were observed after 48 hours and72 hours.

In the culture receiving 0.1% of Rid Fungus, sporangia and zoosporeswere observed after 1 hour. Bacteria and zoospores were observed after24 hours, bacteria and a few zoospores with no motility were observedafter 48 hours, and bacteria only were observed after 72 hours. Theresults show that Rid Fungus may be effective for treating chytrids,however the effect on Haematococcus cells needs to be determined if RidFungus is to be used as treatment during culturing.

Example 14

Several concentrations of salt and Rid Fungus (blend of organic herbs)were further investigated as a chytrid treatment, as in Example 13. Apure chytrid culture in a well plate was treated and visually observedunder a microscope to determine the effect. An untreated culture wasused as a control for comparison purposes.

In the control culture, motile dense zoospores were observed after 1 and4.5 hours. From the observation of the control culture, a treatment thatreduces the formation or motility of zoospores may be furtherinvestigated as a viable treatment.

In the culture receiving 5 ppt salt (NaCl), a less dense mass ofzoospores with some being motile but sluggish were observed after 1hour. A mix of non-motile and motile zoospores were observed after 4.5hours.

In the culture receiving 10 ppt salt (NaCl), a mass of zoospores at thesame density as the 5 ppt treatment but with all being non-motile wereobserved after 1 hour. Non-motile zoospores only were observed after 4.5hours.

In the culture receiving 20 ppt salt (NaCl), a mass of zoospores lessdense than mass of the 5 and 10 ppt treatments but with all beingnon-motile were observed after 1 hour. Non-motile zoospores only wereobserved after 4.5 hours. The results show that the high level of saltis effective against chytrids, but the level is above the threshold thathas been shown to negatively affect some strains of Haematococcus duringculturing.

In the culture receiving 0.10% of Rid Fungus, a mass of zoospores lessdense than the control but highly motile were observed after 1 hour.Motile zoospores were observed after 4.5 hours.

In the culture receiving 0.25% of Rid Fungus, a mass of zoospores lessdense than the control but less motility than the 0.1% treatment wereobserved after 1 hour. Some motile zoospores and possibly dead sporangiawere observed after 4.5 hours.

In the culture receiving 0.50% of Rid Fungus, some motile zoospores wereobserved after 1 hour. Some motile zoospores and dead sporangia wereobserved after 4.5 hours. The results show that the higher levels of RidFungus may be effective in reducing chytrid zoospores, but the effect onHaematococcus cells needs to be determined if Rid Fungus is to be usedas treatment during culturing.

Example 15

Several concentrations of bleach and Lufenuron were further investigatedfor their effectiveness as chytrid treatments for Haematococcuspluvialis during culturing. Cultures of Haematococcus pluvialis(Strain 1) red cyst cells infected with chytrids were treated in wellplates (0.5 mL volume) with bleach or Lufenuron to evaluate the effecton the Haematococcus cells if bleach or Lufenuron were to be used as acontamination treatment. A first control culture of Haematococcus redcyst cells absent of chytrids and a second control culture of healthyHaematococcus red cyst cells inoculated with chytrids were used ascomparisons for the treatments. The cultures were treated with bleach ator less than the tolerance level of Haematococcus: 0.01, 0.02 and 0.03mL/L of 12.5% stock concentration bleach (calculated concentration0.00125, 0.0025, and 0.00375 mL/L), or different concentrations ofLufenuron (0.01, 0.1 and 1% of culture volume). The cultures wereobserved under a microscope during the experiment to determineeffectiveness.

In the first control culture, the cells were observed to be healthy redcysts at the time of inoculation. Healthy red cysts and bacteria wereobserved after 24 hours. Healthy red cysts and some clumping wereobserved after 48 hours. Healthy red cysts were observed after 72 hours.

In the second control culture, swimming zoospores and sporangia on redcysts were observed at the time of inoculation. Swimming zoospores andpositive infection of the red cyst cells were observed after 24 hours.Bacteria, swimming zoospores, and positive infection of the red cystcells were observed after 48 hours. Heavy bacteria, few swimmingzoospores, and decreased infection of the red cyst cells were observedafter 72 hours.

In the culture receiving 0.01 mL/L of bleach 12.5% stock concentration(calculated concentration of 0.00125 mL/L, which is a concentrationbelow the tolerance level of Haematococcus), swimming zoospores andpositive infection were observed after 24 and 48 hours. Some swimmingzoospores, high bacteria, and dead sporangia were observed after 72hours.

In the culture receiving 0.02 mL/L of bleach 12.5% stock concentration(calculated concentration of 0.0025 mL/L, which is a concentration belowthe tolerance level of Haematococcus), swimming zoospores and positiveinfection were observed after 24 and 48 hours. Some swimming zoosporesand dead sporangia were observed after 72 hours.

In the culture receiving 0.03 mL/L of bleach 12.5% stock concentration(calculated concentration of 0.00375 mL/L, which is a concentration atthe tolerance level of Haematococcus), swimming zoospores and positiveinfection were observed after 24. Swimming zoospores, positiveinfection, and lysed/dead Haematococcus cells were observed after 48hours. Some swimming zoospores, high bacteria, and active infection wereobserved after 72 hours. Based on the results, the concentrations ofbleach below the tolerance level of Haematococcus were ineffective attreating chytrids.

In the culture receiving 0.01% of Lufenuron, swimming zoospores andpositive infection were observed after 24 and 48 hours. Some swimmingzoospores, Ochromonas (single-celled, motile, golden-brown alga), andactive infection were observed after 72 hours.

In the culture receiving 0.10% of Lufenuron, dead sporangia, someinfection, and active zoospores were observed after 24 and 48 hours.Some swimming zoospores and active infection were observed after 72hours.

In the culture receiving 1.00% of Lufenuron, dead sporangia andzoospores attached to cyst cells were observed after 24 hours.Ochromonas, bacteria, dead sporangia, zoospores attached to cyst cells,and few swimming zoospores were observed after 48 hours. Ochromonas, noactive infection, and no zoospores were observed after 72 hours. Basedon the results, concentration of 1% Lufenuron may be an effectivetreatment for chytrids in a Haematococcus culture, however the effect onbiomass and carotenoid accumulation needs to be determined.

Example 16

A solution comprising 50% sodium hydroxide (NaOH) was evaluated as atreatment for chytrids that could be applied to empty reactors in orderto clean them for subsequent batches. A pure culture of chytridzoospores and sporangia was treated with a 1% dose of a solutioncomprising 50% NaOH at temperatures of 25, 40, and 50° C. The cultureswere then observed under a microscope for a reduction in the chytridzoospores or sporangia number, or a reduction in the integrity of thechytrid cells. The results showed that there was not a reduction inchytrid zoospores or sporangia number or integrity for any of thetreatments.

Example 17

Experiments were conducted to evaluate the use of biological agents forthe control of chytrids in a culture of microalgae. Ochromonas is asingle-celled, motile, golden-brown alga known to ingest bacteria andsmall eukaryotes, and was evaluated for its properties as a chytridzoospore predator. Janthinobacterium is a gram-negative soil bacteriaknown to prey on chytrid zoospores and was evaluated for its propertiesas such. A pure culture of chytrids was inoculated into well plates. Acontrol was left untreated and compared to cultures treated withOchromonas and Janthinobacterium. Chytrid zoospore density wasquantified after three days by visual observation under a microscope.Results showed that the culture treated with Ochromonas had an averagenumber of zoospores that was approximately half of the culture treatedwith Janthinobacterium and approximately one third of the control.Further testing would have to be performed to determine the effect onHaematococcus cells before use as culturing treatment.

Aspects of the Invention

In one non-limiting embodiment of the invention, a method of culturingHaematococcus pluvialis, may comprise: culturing a population ofHaematococcus pluvialis cells in growth conditions in a liquid culturemedium to obtain a culture of Haematococcus pluvialis cells in which thecells are primarily in a green swimmer stage; contacting the primarilygreen swimmer stage culture with hydrogen peroxide to form a calculatedconcentration in the range of 0.005-0.020 mL of hydrogen peroxide per Lof culture medium (mL/L); and culturing the Haematococcus pluvialiscells in reddening conditions to form cells in the red cyst stage foraccumulation of carotenoids.

In some embodiments, the calculated concentration of hydrogen peroxidemay be in the range of 0.005-0.010 mL/L. In some embodiments, thecalculated concentration of hydrogen peroxide may be in the range of0.010-0.015 mL/L. In some embodiments, the calculated concentration ofhydrogen peroxide is in the range of 0.015-0.020 mL/L.

In some embodiments, the growth conditions may comprise aphotosynthetically active radiation intensity in the range of 30-60 molm⁻² d⁻¹, nitrate concentration in the range of 20-50 ppm in the culturemedium, and less than 1 ppt of sodium chloride in the culture medium. Insome embodiments, the reddening conditions may comprise the present of1-5 ppt sodium chloride in the culture medium.

In some embodiments, the method may further comprise determining a levelof chytrids in the culture of Haematococcus pluvialis cells as apercentage of infected cells out of the total cells in a culture. Insome embodiments, the culture of Haematococcus pluvialis cells may becontacted with the hydrogen peroxide when the level of chytrids is lessthan 20%. In some embodiments, the culture of Haematococcus pluvialiscells is contacted with the hydrogen peroxide when the level of chytridsis at least 5%.

In some embodiments, the level of chytrids in the culture may bemaintained below the level of chytrids at the time of contact withhydrogen peroxide while culturing the Haematococcus pluvialis cells inreddening conditions to produce cells in the red cyst stage for theaccumulation of carotenoids. In some embodiments, the chytrid levelafter contacting the culture with hydrogen peroxide may be 20-95% lessthan a control culture not receiving treatment with hydrogen peroxide.

In some embodiments, the cells may be contacted with the hydrogenperoxide multiple times. In some embodiments, the cells may be contactedwith the hydrogen peroxide every 6-24 hours. In some embodiments, thecells may be contacted with the hydrogen peroxide every 6-12 hours. Insome embodiments, the cells may be contacted with the hydrogen peroxideevery 6-8 hours. In some embodiments, the cells may be contacted withthe hydrogen peroxide every day over the course of 1-14 days. In someembodiments, the cells may be contacted with hydrogen peroxide everyother day over the course of 3-15 days.

In some embodiments, the biomass yield of the Haematococcus pluvialiscells contacted with the hydrogen peroxide may be equivalent to orgreater than a control culture not receiving treatment with hydrogenperoxide. In some embodiments, the biomass yield of the Haematococcuspluvialis cells contacted with the hydrogen peroxide may be 0.01-0.25g/L greater than a control culture not receiving treatment with hydrogenperoxide.

In some embodiments, the carotenoids yield of the Haematococcuspluvialis cells contacted with the hydrogen peroxide may be equivalentto or greater than a control culture not receiving treatment withhydrogen peroxide. In some embodiments, the carotenoid yield of theHaematococcus pluvialis cells contacted with the hydrogen peroxide maybe 0.10-1.50% greater than a control culture not receiving treatmentwith hydrogen peroxide.

In some embodiments, the method may further comprise transferring theculture of Haematococcus pluvialis cells to a new culturing vessel aftercontacting the culture with the hydrogen peroxide.

In one non-limiting embodiment of the invention, a method of culturingHaematococcus pluvialis may comprise: culturing a population ofHaematococcus pluvialis cells in reddening conditions in a liquidculture medium comprising 1-5 ppt of salt to obtain a culture ofHaematococcus pluvialis cells in which the cells are primarily in acyst; and contacting the primarily cyst stage culture with hydrogenperoxide to form a calculated concentration in the range of 0.005-0.020mL of hydrogen peroxide per L of culture medium (mL/L). In someembodiments, the cyst stage may comprise at least one selected from thegroup consisting of green cysts and red cysts accumulating carotenoids.

In some embodiments, the salt may be sodium chloride. In someembodiments, the sodium chloride may be present in the liquid culturemedium at a concentration of 1-3 ppt. In some embodiments, the sodiumchloride may be present in the liquid culture medium at a concentrationof 1-2 ppt.

In some embodiments, the chytrid level after contacting the culture withthe hydrogen peroxide may be 10-95% less than a control culture notreceiving treatment with hydrogen peroxide. In some embodiments, thebiomass yield of the Haematococcus pluvialis cells contacted with thehydrogen peroxide may be 0.01-0.30 g/L greater than a control culturenot receiving treatment with hydrogen peroxide.

In one non-limiting embodiment of the invention, a method of culturingHaematococcus pluvialis may comprise: culturing a population ofHaematococcus pluvialis cells in reddening conditions in a liquidculture medium to obtain a culture of Haematococcus pluvialis cells inwhich the cells are primarily in a cyst stage; detecting a presence ofchytrids in the culture; and contacting the culture comprising chytridsand red cyst cells with 5-20 ppt salt. In some embodiments, the cyststage may comprise at least one selected from the group consisting ofgreen cysts and red cysts accumulating carotenoids.

In some embodiments, the salt may be sodium chloride. In someembodiments, the concentration of sodium chloride in the culture mediummay be in the range of 5-10 ppt. In some embodiments, the concentrationof sodium chloride in the culture medium may be in the range of 10-15ppt. In some embodiments, the concentration of sodium chloride in theculture medium may be in the range of 15-20 ppt.

In some embodiments, the culture of Haematococcus pluvialis cells may becontacted with the salt when a level of cells infected by chytrids isless than 20% of the total cells. In some embodiments, the culture ofHaematococcus pluvialis cells may be contacted with salt when a level ofcells infected by chytrids is at least 5% of the total cells.

In some embodiments, the level of chytrids in the culture may bemaintained below the level of chytrids at the time of contact with thesalt while culturing the Haematococcus pluvialis cells in reddeningconditions to form cells in the red cyst stage for the accumulation ofcarotenoids.

In one non-limiting embodiment of the invention, a method of preventinga chytrid infection in a culture of Haematococcus pluvialis maycomprise: culturing a population of Haematococcus pluvialis cells in aliquid culture medium; determining a number of Haematococcus pluvialiscells infected with chytrids in the culture; contacting the culture withhydrogen peroxide when the percentage of Haematococcus pluvialis cellsinfected with chytrids is less than 10% of the total cells; continuingto culture the Haematococcus pluvialis cells; and verifying that apercentage of Haematococcus pluvialis cells infected with chytrids isless than 10% of the total cells after contact with the hydrogenperoxide.

In one non-limiting embodiment, a method of culturing Haematococcuspluvialis may comprise: culturing a population of Haematococcuspluvialis cells in a liquid culture medium in growth conditions toobtain a culture of Haematococcus pluvialis cells in which the cells areprimarily in a green swimmer stage; contacting the primarily greenswimmer cell stage culture with hydrogen peroxide to form a calculatedconcentration in the range of 0.005-0.025 mL of hydrogen peroxide per Lof culture medium (mL/L) prior to the formation of cell cysts; andcontinuing to culture the Haematococcus pluvialis cells in growthconditions. In some embodiments, the calculated concentration ofhydrogen peroxide may be in the range of 0.005-0.010, 0.010-0.015,0.015-0.020, or 0.020-0.025

In some embodiments, the method may further comprise determining a levelof lysis in the culture of Haematococcus pluvialis cells as a percentageof the total Haematococcus pluvialis cells in the culture. In someembodiments, the culture of Haematococcus pluvialis cells may becontacted with the hydrogen peroxide when the level of lysis is lessthan 20%. In some embodiments, the culture of Haematococcus pluvialiscells may be contacted with the hydrogen peroxide when the level oflysis is less than 5%.

In some embodiments, the level of lysis in the culture may be maintainedat or below the level of lysis at the time of contact with the hydrogenperoxide while continuing to culture the Haematococcus pluvialis cellsin growth conditions. In some embodiments, the lysis level of theHaematococcus pluvialis culture after contact with the hydrogen peroxidemay be 1-80% less than a lysis level in a control culture not receivingtreatment with hydrogen peroxide.

In some embodiments, the method may further comprise determining a livebacteria count in the culture of Haematococcus pluvialis cells. In someembodiments, the live bacteria count may be reduced 10-15×10⁵ CFU/mLafter contact with the hydrogen peroxide. In some embodiments, the livebacteria count may be maintained below 10⁷ CFU/mL following contact withthe hydrogen peroxide.

In one non-limiting embodiment, a method of preventing lysis in aculture of Haematococcus pluvialis may comprise: culturing a populationof Haematococcus pluvialis cells in a liquid culture medium in growthconditions to obtain a culture of Haematococcus pluvialis cells in whichthe cells are primarily in a green swimmer stage; determining a level ofcell lysis for the Haematococcus pluvialis cells; contacting theprimarily green swimmer cell stage culture with hydrogen peroxide priorto the formation of cysts when the lysis level of Haematococcuspluvialis cells is less than 5%; continuing to culture the Haematococcuspluvialis cells in growth conditions; and verifying that the level oflysis of Haematococcus pluvialis cells is less than 5% after contactwith the hydrogen peroxide.

In one non-limiting embodiments, a microalgae culture composition maycomprise: a population of Haematococcus pluvialis cells in a liquidculture medium; and a calculated concentration of hydrogen peroxide inthe range of 0.005-0.025 mL of hydrogen peroxide per L of culture medium(mL/L), wherein hydrogen peroxide has been added to the culture mediumin the previous 120 minutes.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Allprovided ranges of values are intended to include the end points of theranges, as well as values between the end points.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having,” “including,” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention. Any claimed embodiment of the invention does notnecessarily include all of the “aspects” or “embodiments” of thespecification.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subjectmatter recited in the claims and/or aspects appended hereto as permittedby applicable law.

What is claimed is:
 1. A method of culturing Haematococcus pluvialis,comprising: a. Culturing a population of Haematococcus pluvialis cellsin a liquid culture medium in growth conditions to obtain a culture ofHaematococcus pluvialis cells in which the cells are primarily in agreen swimmer stage; b. Contacting the primarily green swimmer cellstage culture with hydrogen peroxide to form a calculated concentrationin the range of 0.005-0.025 mL of hydrogen peroxide per L of culturemedium (mL/L) prior to the formation of cell cysts; and c. Continuing toculture the Haematococcus pluvialis cells in growth conditions.
 2. Themethod of claim 1, wherein the calculated concentration of hydrogenperoxide is in the range of 0.005-0.010 mL/L.
 3. The method of claim 1,wherein the calculated concentration of hydrogen peroxide is in therange of 0.010-0.015 mL/L.
 4. The method of claim 1, wherein thecalculated concentration of hydrogen peroxide is in the range of0.015-0.020 mL/L.
 5. The method of claim 1, wherein the calculatedconcentration of hydrogen peroxide is in the range of 0.020-0.025 mL/L.6. The method of claim 1, wherein the growth conditions comprise aphotosynthetically active radiation intensity below 30-60 mol m⁻² d⁻¹,nitrates in the range of 20-50 ppm in the culture medium, and less than1 ppt of sodium chloride in the culture medium.
 7. The method of claim1, wherein the method further comprises: a. Determining a level of lysisin the culture of Haematococcus pluvialis cells as a percentage of cellsdisplaying a loss of cell membrane integrity out of the totalHaematococcus pluvialis cells in the culture.
 8. The method of claim 7,wherein the culture of Haematococcus pluvialis cells is contacted withthe hydrogen peroxide when the level of lysis is less than 20%.
 9. Themethod of claim 8, wherein the culture of Haematococcus pluvialis cellsis contacted with the hydrogen peroxide when the level of lysis is lessthan 5%.
 10. The method of claim 7, wherein the level of lysis in theculture is maintained at or below the level of lysis at the time ofcontact with the hydrogen peroxide while continuing to culture theHaematococcus pluvialis cells in growth conditions.
 11. The method ofclaim 10, wherein the level of lysis of the Haematococcus pluvialisculture after contact with the hydrogen peroxide is 1-80% less than alevel of lysis in a control culture not receiving treatment withhydrogen peroxide.
 12. The method of claim 1, wherein the cells arecontacted with the hydrogen peroxide multiple times.
 13. The method ofclaim 12, wherein the cells are contacted with the hydrogen 2-4 timesper day.
 14. The method of claim 12, wherein the cells are contactedwith the hydrogen peroxide every day over the course of 1-14 days. 15.The method of claim 12, wherein the cells are contacted with thehydrogen peroxide every other day for a period of 3-15 days.
 16. Themethod of claim 1, wherein the biomass yield of the Haematococcuspluvialis cells contacted with hydrogen peroxide is equivalent to orgreater than a control culture not receiving treatment with hydrogenperoxide.
 17. The method of claim 16, wherein the biomass yield of theHaematococcus pluvialis cells contacted with hydrogen peroxide is0.01-0.25 g/L greater than a control culture not receiving treatmentwith hydrogen peroxide.
 18. The method of claim 1, wherein the methodfurther comprises: a. Determining a live bacteria count in the cultureof Haematococcus pluvialis cells.
 19. The method of claim 18, whereinthe live bacteria count is reduced 10-15×10⁵ CFU/mL after contact withthe hydrogen peroxide.
 20. The method of claim 18, wherein the livebacteria count is maintained below 10⁷ CFU/mL following contact with thehydrogen peroxide.